PSMA-targeted NIR dyes and their uses

ABSTRACT

The present disclosure relates to prostate specific membrane antigen (PSMA) targeted compounds conjugated to near-infra red (NIR) dyes and methods for their therapeutic and diagnostic use. More specifically, this disclosure provides compounds and methods for diagnosing and treating diseases associated with cells and/or vasculature expressing prostate specific membrane antigen (PSMA), such as prostate cancer and related diseases. The disclosure further describes methods and compositions for making and using the compounds, methods incorporating the compounds, and kits incorporating the compounds.

RELATED APPLICATIONS

The present patent application is a continuation of U.S. patentapplication Ser. No. 14/939,915, which was filed on Nov. 12, 2015, whichis a continuation of U.S. patent application Ser. No. 14/937,169, whichwas filed on Nov. 10, 2015 and claimed the priority benefit of U.S.Provisional Patent Application Ser. No. 62/216,157, filed Sep. 9, 2015the content of which is hereby incorporated by reference in its entiretyinto this disclosure.

FIELD OF THE INVENTION

The present disclosure relates to prostate specific membrane antigen(PSMA)-targeted near-infra red (NIR) dyes and methods for theirtherapeutic and diagnostic use. More specifically, this disclosureprovides compounds and methods for diagnosing and surgical removal(image-guided surgery) of cells expressing prostate specific membraneantigen (PSMA), such as prostate cancer and related diseases. Thedisclosure further describes methods and compositions for making andusing the compounds, methods incorporating the compounds, and kitsincorporating the compounds.

BACKGROUND OF THE INVENTION

The prostate is one of the male reproductive organs found in the pelvisbelow the urinary bladder. It functions to produce and store seminalfluid which provides nutrients and fluids that are vital for thesurvival of sperm introduced into the vagina during reproduction. Likemany other tissues, the prostate glands are also prone to develop eithermalignant (cancerous) or benign (non-cancerous) tumors. The AmericanCancer Society predicted that over 230,000 men would be diagnosed withprostate cancer and over 30,000 men would die from the disease in year2005. In fact, prostate cancer is one of the most common male cancers inwestern societies, and is the second leading form of malignancy amongAmerican men. Current treatment methods for prostate cancer includehormonal therapy, radiation therapy, surgery, chemotherapy, photodynamictherapy, and combination therapy. The selection of a treatment generallyvaries depending on the stage of the cancer. However, many of thesetreatments affect the quality of life of the patient, especially thosemen who are diagnosed with prostate cancer over age 50. For example, theuse of hormonal drugs is often accompanied by side effects such asosteoporosis and liver damage. Such side effects might be mitigated bythe use of treatments that are more selective or specific to the tissuebeing responsible for the disease state, and avoid non-target tissueslike the bones or the liver. As described herein, prostate specificmembrane antigen (PSMA) represents a target for such selective orspecific treatments.

Surgical removal of malignant disease constitutes one of the most commonand effective therapeutic for primary treatment for cancer. Resection ofall detectable malignant lesions results in no detectable return of thedisease in approximately 50% of all cancer patients' and may extend lifeexpectancy or reduce morbidity for patients in whom recurrence of thecancer is seen. Not surprisingly, surgical methods for achieving morequantitative cytoreduction are now receiving greater scrutiny.

Resection of all detectable malignant lesions results in no detectablereturn of the disease in approximately 50% of all cancer patients andmay extend life expectancy or reduce morbidity for patients in whomrecurrence of the cancer is seen. Given the importance of totalresection of the malignant lesions, it is beneficial to ensure that themalignant lesions are accurately and completely identified.Identification of malignant tissue during surgery is currentlyaccomplished by three methods. First, many tumor masses and nodules canbe visually detected based on abnormal color, texture, and/ormorphology. Thus, a tumor mass may exhibit variegated color, appearasymmetric with an irregular border, or protrude from the contours ofthe healthy organ. A malignant mass may also be recognized tactilely dueto differences in plasticity, elasticity or solidity from adjacenthealthy tissues. Finally, a few cancer foci can be locatedintraoperatively using fluorescent dyes that flow passively from theprimary tumor into draining lymph nodes. In this latter methodology,fluorescent (sentinel) lymph nodes can be visually identified, resectedand examined to determine whether cancer cells have metastasized tothese lymph nodes.

PSMA is named largely due to its higher level of expression on prostatecancer cells; however, its particular function on prostate cancer cellsremains unresolved. PSMA is over-expressed in the malignant prostatetissues when compared to other organs in the human body such as kidney,proximal small intestine, and salivary glands. PSMA also express in theneo-vasculature of most of the solid tumors. Though PSMA is expressed inbrain, that expression is minimal, and most ligands of PSMA are polarand are not capable of penetrating the blood brain barrier. PSMA is atype II cell surface membrane-bound glycoprotein with −110 kD molecularweight, including an intracellular segment (amino acids 1-18), atransmembrane domain (amino acids 19-43), and an extensive extracellulardomain (amino acids 44-750). While the functions of the intracellularsegment and the transmembrane domains are currently believed to beinsignificant, the extracellular domain is involved in several distinctactivities. PSMA plays a role in central nervous system, where itmetabolizes N-acetyl-aspartyl glutamate (NAAG) into glutamic andN-acetyl aspartic acid. Accordingly, it is also sometimes referred to asan N-acetyl alpha linked acidic dipeptidase (NAALADase). PSMA is alsosometimes referred to as a folate hydrolase I (FOLH I) or glutamatecarboxypeptidase (GCP II) due to its role in the proximal smallintestine where it removes γ-linked glutamate from poly-y-glutamatedfolate and a-linked glutamate from peptides and small molecules.

PSMA also shares similarities with human transferrin receptor (TfR),because both PSMA and TfR are type II glycoproteins. More specifically,PSMA shows 54% and 60% homology to TfR1 and TfR2, respectively. However,though TfR exists only in dimeric form due to the formation ofinter-strand sulfhydryl linkages, PSMA can exist in either dimeric ormonomeric form.

Unlike many other membrane-bound proteins, PSMA undergoes rapidinternalization into the cell in a similar fashion to cell surface boundreceptors like vitamin receptors. PSMA is internalized throughclathrin-coated pits and subsequently can either recycle to the cellsurface or go to lysosomes. It has been suggested that the dimer andmonomer form of PSMA are inter-convertible, though direct evidence ofthe interconversion is being debated. Even so, only the dimer of PSMApossesses enzymatic activity, and the monomer does not.

Though the role of the PSMA on the cell surface of the prostate cancercells remains unknown, it has been recognized that PSMA represents aviable target for the selective and/or specific delivery of biologicallyactive agents, including diagnostic agents, imaging agents, andtherapeutic agents to such prostate cancer cells.

The radio-immunoconjugate of the anti-PSMA monoclonal antibody (mAb)7E11, known as the PROSTASCINT® scan, is currently being used todiagnose prostate cancer metastasis and recurrence. However, this agenttends to produce images that are challenging to interpret (Lange, P. H.PROSTASCINT scan for staging prostate cancer. Urology 2001, 57, 402-406;Haseman, M. K.; et al. Cancer Biother Radiopharm 2000, 15, 131-140;Rosenthal, S. A.; et al. Tech Urol 2001, 7, 27-37). It binds to anintracellular epitope of PSMA in necrotic prostate cancer cells. Morerecently, monoclonal antibodies have been developed that bind to theextracellular domain of PSMA and have been radiolabeled and shown toaccumulate in PSMA-positive prostate tumor models in animals. However,diagnosis and tumor detection using monoclonal antibodies has beenlimited by the low permeability due to their large size (150,000 Da) andslow clearance from non-targeted tissue. Moreover, the selectivetargeting of radio—or optical imaging agents either for imaging ortherapeutic purposes is challenging due to their long half-life (˜30days). Especially, patients have to be stay in the hospital for longerdays and spend more money on medical bills.

Two promising approaches to fluorescence-guided surgery are currentlyunder intense investigation for use in the clinic. In one method, anactivatable NIR fluorescent probe, which is minimally fluorescent in thesteady state due to its proximity to an attached quencher, becomeshighly fluorescent upon release of the quencher in malignant tissue. Oneof the most commonly used release mechanisms involves incorporation of apeptide sequence between the dye and the quencher that can bespecifically cleaved by a tumor-enriched protease (i.e. cathepsins,caspases and matrix metalloproteinases). A major advantage of thisstrategy lies in the absence of fluorescence in tissues that lack theactivating enzyme, allowing tissues along the excretion pathway (e.g.kidneys, bladder, liver) to remain nonfluorescent unless theyfortuitously express the cleaving enzyme. Such tumor-activated NIR dyescan also generate substantial fluorescence in the tumor mass as long asthe malignant lesion is enriched in the cleaving protease and thereleased dye is retained in the tumor. The major disadvantage of thismethodology arises from the poor tumor specificities of many of therelevant hydrolases (most of which are also expressed in healthy tissuesundergoing natural remodeling or experiencing inflammation). Moreover,the abundance of the desired proteases may vary among tumor masses,leading to slow or no activation of fluorescence in some malignantlesions and rapid development of fluorescence in others. Most of thetime, these activatable peptides contain over 20 amino acids linked viapeptide bonds that could lead to higher molecular weights, longer leadtime (24 h), cleavage of peptide bonds by peptidase in the circulation,high false positive results and very high manufacturing costs.

Other release mechanisms that activatable dyes use are pH differencebetween circulation and within the tumor or change in redox potential.

In the second, a fluorescent dye is conjugated to a tumor-specifictargeting ligand that causes the attached dye to accumulate in cancersthat over-express the ligand's receptor. While PSMA-targetedantibody-NIR dye conjugates have not yet been entered to clinical trialsfor fluorescence-guided surgery of cancer, several types of NIR dyeshave been conjugated to monoclonal antibodies such as Her-2 with theintent of clinical development. Unfortunately, most of these dyes aretethered to antibodies non-specifically via amide, disulfide, ormaleimide chemistry using either lysine or cysteine residues in theprotein leading to heterogeneous chemical entities which result invariable affinities, efficacies, PK and safety profiles. Moreover,maleimide and disulfide bonds are known to be unstable in thecirculation (half-life-≤2 h). On the other hand, lack of precisestructural definition may limit progression of these conjugates into theclinical use due to challenges associated with the production processand safety. Moreover, production of these antibodies is highly expensivewhen compared to small molecular ligands. In contrast, small moleculeligand (Mr>0.5 Da), can penetrate solid tumors rapidly, and clears fromPSMA-negative tissues in <2 h, shows high tumor-to-background ratios,easy of synthesis, and stable during the synthesis and storage.

Despite all the advantages those small molecular ligands have,development of NIR dye that maintains or enhances the properties of thesmall molecule is challenging. Recently, a variety of low molecularweight inhibitors of PSMA have been conjugated to visible light wavelength dyes (400-600 nm) such as fluorescein and rhodamine and tested inin animal models [Kularatne S A, Wang K, Santhapuram H K, Low P S. MolPharm. 2009 May-June; 6(3):780-9] or in cells in culture [Liu T,Nedrow-Byers J R, Hopkins M R, Berkman C E. Bioorg Med Chem Lett. 2011Dec. 1; 21(23)] or in human blood samples (He W, Kularatne S A, Kalli KR, Prendergast F G, Amato R J, Klee G G, Hartmann L C, Low P S. Int JCancer. 2008 Oct. 15; 123(8):1968-73).

The visible light wave length dyes are not optimal for intra-operativeimage-guided surgery as these dyes are associated with a relatively highlevel of nonspecific background light due to the presence of collagen inthe tissues. Hence the signal to noise ratio from these conventionalcompounds is low. Moreover, the absorption of visible light bybiological chromophores, in particular hemoglobin, limits thepenetration depth to a few millimeters. Thus tumors that are burieddeeper than a few millimeters in the tissue typically remain undetected.Furthermore ionization equilibrium of fluorescein (pKa=6.4) leads topH-dependent absorption and emission over the range of 5 to 9.Therefore, the fluorescence of fluorescein-based dyes is quenched at lowpH (below pH 5).

Therefore, NIR dyes conjugated to small molecule ligands that targetPSMA [(a) Humblet V, Lapidus R, Williams L R, Tsukamoto T, Rojas C,Majer P, Hin B, Ohnishi S, De Grand A M, Zaheer A, Renze J T, NakayamaA, Slusher B S, Frangioni J V. Mol Imaging. 2005 October-December;4(4):448-62.; (b) Thomas M, Kularatne S A, Qi L, Kleindl P, Leamon C P,Hansen M J, Low P S.; (c) Chen Y, Dhara S, Banerjee S R, Byun Y,Pullambhatla M, Mease R C, Pomper M G. Biochem Biophys Res Commun. 2009Dec. 18; 390(3):624-9; (d) Nakajima T, Mitsunaga M, Bander N H, Heston WD, Choyke P L, Kobayashi H. Bioconjug Chem. 2011 Aug. 17; 22(8):1700-5.;(e) Chen Y, Pullambhatla M, Banerjee S R, Byun Y, Stathis M, Rojas C,Slusher B S, Mease R C, Pomper M G. Bioconjug Chem. 2012 Dec. 19;23(12):2377-85.; (f) Laydner H, Huang S S, Heston W D, Autorino R, WangX, Harsch K M, Magi-Galluzzi C, Isac W, Khanna R, Hu B, Escobar P,Chalikonda S, Rao P K, Haber G P, Kaouk J H, Stein R J. Urology. 2013February; 81(2):451-6.; (g) Kelderhouse L E, Chelvam V, Wayua C,Mahalingam S, Poh S, Kularatne S A, Low P S. Bioconjug Chem. 2013 Jun.19; 24(6):1075-80.] have been tested as imaging agents in murine modelsof prostate cancer.

While these PSMA-targeted NIR dyes showed some labeling of prostatecancer cells in culture, they had very weak fluorescence inPSMA-expressing prostate tumor xenograft animal models. For example, themolecules described by, Humblet et al have shown very low tumoraccumulation and florescence in the tumor xenograft models. It may bedue the lack of proper spacer between the ligand the NIR dye may havehindered the binding of ligand to the binding pocket in PSMA. On theother hand, phosphorous based ligands have less affinity for PSMA whencompared to DUPA. Moreover, phosphorous based ligands are difficult tosynthesize, involve multiple steps, and will be expensive tomanufacture.

PSMA-targeted NIR agent reported in Chen et al has taken over 20 h toreach the tumor and 72 h clear from the non-targeted tissues. Alsonotably, this PSMA-targeted NIR dye has very slowly skin clearance.While binding epitope of PSMA in transfected cells that they used can beartificial, it had very low uptake and low fluorescence in PSMAtransfected prostate cancer cell tumor. Furthermore, there issubstantial non-specific uptake of this molecule in all other tissuesand there is accumulation and fluorescence in PSMA-negative cellsindicating non-specific and non-targeted nature of NIR conjugatereported by Chen et al.

Chen et al and Laydner et al also have conjugated a small moleculeligand to IR800CW (a NIR dye). IR800CW is asymmetrical dye withactivated carboxylic acid with n-hydroxysuccinimide ester (NHS). This isan extremely expensive molecule to synthesize and even more to purchasefrom commercially available resources (1 g is over $60,000). IR800CWalso has the disadvantage that it is not stable during the synthesis dueto two reasons: (a) hydrolysis of NHS ester, (b) hydrolysis of vinylether. The lack of stability of IR800CW conjugates during synthesisleads to formation of over 60% of undesired byproducts. This requirescomplex purification techniques indicating path for higher productioncost, higher waiting period for clinical translation, and surgeons andpatients will not have access to the drug.

Laydner et al conjugated a PSMA ligand to IR800CW via a long peptidespace (6 amino acids) and bifunctional linker with NHS and maleimide. Inaddition to all the disadvantages caused by IR800CW, this PSMA-targetedIR800CW conjugate has a complicated synthesis scheme requiring synthesisin five stages (synthesis of ligand, conjugation of ligand tobifunctional linker via maleimide functional group, synthesis of peptidelinker, conjugation of peptide linker to IR800CW, conjugation of peptidelinker-IR800CW to ligand-bifunctional linker via amide bond) in multiplesteps. Therefore, the manufacturing costs hamper the effectiveproduction of this molecule for clinical purposes. The synthesis schemefor these molecules is further complicated due to multiple chiralcenters in the molecule. Peptide spacers, however, possess multiplechiral centers (stereoisomers) typically necessitating the need forproduction and assessment of all stereoisomers for FDA clearance. Forexample, a peptide spacer possessing only 3 amino acids (i.e. 3 chiralcenters), would require toxicity profiles for 8 different drug productssince these heterogeneous mixtures could result in different affinities,efficacies, PK and safety profiles.

The small molecule ligand used by Laydner et al is GluNHCONHCys-SH. Thefree thiol moiety in Cys tends to oxidize hence the molecule has to behandled under argon or nitrogen environment and generally leads to anunstable molecule. GluNHCONHCys-SH ligand is conjugated to bifunctionallinker via maleimide reaction. It is well reported that reactionsbetween thiols and maleimide are reversible and yield 50% of thediseased product. Moreover, maleimide bonds are not stable incirculation in the human body, hence use of maleimide bonds risk therelease of the non-targeted dye leading to non-specific uptake thereof.

Kelderhouse et al conjugated DUPA-linker-Cys to Alexa flour 647 andDylight 750 to DUPA via maleimide group. Again, these molecules have allthe disadvantages associated with maleimide. Moreover, these low wavelength NIR dyes, while being commercially available are very expensive.While molecules were tested on experimental metastatic mouse model,images were inconclusive.

Liu et al also reported PSMA-targeted NIR dye and some in vitro data butno animal data were reported. The lack of a proper spacer between theligand and the NIR dye may have attributed to the lack of vivo data.Moreover, this dye has many drawbacks as other reported compounds. It isa phosphorous based ligand and asymmetrical dye. So, it hasdisadvantages described of both phosphorous based ligands as well asasymmetrical NIR dyes.

Nakajima et al reported anti-PSMA antibody (J591) conjugated to ICG.Unfortunately, this compound took 72 hours to clear from the otherhealthy tissues such as liver. In addition, the compound remained incirculation for 6 days indicating that it will remain the body for over30 days in human body. Moreover, ICG was tethered to J591non-specifically via amide using either lysine residues in the proteinleading to heterogeneous chemical entities which result in variableaffinities, efficacies, PK and safety profiles. Lack of precisestructural definition may limit progression of these conjugates forclinical use due to challenges associated with the production processand safety.

Higher non-specificity and slow clearance from the skin of reportedPSMA-targeted NIR dyes may be due to poor pharmacokinetic (PK)properties of these compounds.

Thus, there remains a need for a dye substance that can be used tospecifically target PSMA expressing cancer cells or neo-vasculature ofdiseased tissue with increased stability, better PK properties, highersolubility, fast tumor accumulation, high fluorescence, fast skinclearance, and higher tumor-to-background ratios (TBR) for use in vivotissue imaging and to use in image-guided surgery.

BRIEF SUMMARY OF THE INVENTION

This disclosure provides PSMA-targeted ligands linked to NIR dyes viadifferent linkers to improve clinical properties (e.g. stability, PKproperties, solubility, fast tumor accumulation, higher fluorescence,fast skin clearance, and higher tumor-to-background ratios) of thecompounds. The disclosure provides uses of the compounds in image-guidedsurgery and methods for synthesizing the same. This disclosure furtherprovides variation of the total charge of the Ligand-Linker-NIR dyeconjugate by adding positive charges to the linker or reducing number ofnegative charges in the dye molecules. This disclosure also providesnovel higher affinity ligands to improve in vivo affinity and PKproperties of NIR conjugates. This disclosure also provides compoundsfor use in the targeted imaging of tumors expressing PSMA, including butnot limited to prostate cancer, and methods of use, for example, inimaging and surgery involving PSMA positive tissues and tumors.

In certain embodiments, compounds of the present invention have theform: B-X-Y-Z

-   -   wherein B is a PSMA-targeted molecule;    -   X is a spacer;    -   Y is an amino acid spacer; and    -   Z is a NIR dye.

In some embodiments, the PSMA-targeted molecule is chosen from the groupconsisting of a small molecule, a ligand, an inhibitor, an agonist or aderivative thereof. In some embodiments, the PSMA-targeted compound is aligand. In some embodiments, the PSMA-targeted compound is DUPA. Inother embodiments, the PSMA-targeted compound is a small molecule thatbinds PSMA.

In some embodiments, X is a hydrophobic spacer. In some embodiments, Xis selected from the group consisting of an eight aminooctonoic acid(EAOA), a chain of 7 atoms, a spacer 7 atoms in length, a chain from 7to 24 atoms in length; a peptide comprising two aryl or aryl alkylgroups, each of which is optionally substituted, and where one aryl oraryl alkyl group is about 7 to about 11, or about 7 to about 14 atoms,and the other aryl or aryl alkyl group is about 10 to about 14, or about10 to about 17 atoms. In another embodiment, the spacer ccomprises about1 to about 30 atoms, or about 2 to about 20 atoms. In some embodiments,the spacer is 7 atoms in length. In some embodiments, the spacercomprises EAOA. In some embodiments, the spacer is variably charged. Insome embodiments, X has a positive charge. In other embodiments, X has anegative charge.

In some embodiments, Y is selected from the group consisting of: acidic(negatively charged) amino acids, such as aspartic acid and glutamicacid and derivative thereof; basic (positively charged) amino acids suchas arginine, histidine, and lysine and derivative thereof; neutral polaramino acids, such as glycine, serine, threonine, cysteine, tyrosine,asparagine, and glutamine and derivative thereof; neutral nonpolar(hydrophobic) amino acids, such as alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine; and derivativesthereof. In some embodiments, Y is an aromatic amino acid and derivativethereof. In some embodiments, Y has a positive charge. In otherembodiments, Y has a negative charge.

In some embodiments, Z is selected from the group consisting ofnear-infra red dyes, including but not limited to, LS288, IR800, SP054,S0121, KODAK, S2076, S0456 and/or the dyes selected from groupconsisting of:

In certain embodiments, the Z is variably charged. In some embodiments,Z has a positive charge. In other embodiments, Z has a negative charge.

In certain embodiments, compounds of the present invention have theformula:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Y is an amino acidspacer with a sulfur-containing side chain group; and Z is an NIR dye.In some embodiments, the amino acid spacer with a sulfur-containing sidegroup is cysteine. In some embodiments, the amino acid spacer with asulfur-containing side group is methionine. In some embodiments, theamino acid spacer with a sulfur-containing side group is moleculecontaining thiophenol moiety.

In some embodiments, compounds of the present invention have the form:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Y is an amino acidspacer with a chalcogen-containing side chain group; and Z is an NIRdye.

In some embodiments the present invention provides compounds of theform:B-X-Y-ZWherein, B is a PSMA-targeted compound; X is a spacer; Y is an aminoacid chosen from the group consisting of tyrosine, cysteine, lysine, ora derivative thereof; and Z is an NIR dye. In some embodiments, Ycomprises a tyrosine or tyrosine derivative. In some embodiments, Ycomprises a tyrosine and a carbon isotope is on the aromatic ring oftyrosine. In some embodiments, Y comprises an amino acid with anaromatic ring with a hydrogen isotope. In some embodiments the inventionincludes the compound B-X-Y-Z wherein B comprises DUPA or a derivativethereof, X comprises an EAOA, Y comprises tyrosine, and Z comprisesS0456.

The present invention also relates to a compound having the structuralformula:

-   -   or a pharmaceutically acceptable salt thereof, or isotopes        thereof, wherein:    -   R₁ represents a hydrogen or SO₃H;    -   R₂ represents a hydrogen, CH₃, C₃H₆SO₃ ⁻, C₃H₆SO₃H or C₄H₈SO₃ ⁻,        or C₄H₈SO₃H or C₃H₆N⁺(CH₃)₃;    -   R₃, and R₅ each represents a carbon, optionally one or more        sharing bonds,    -   R₄ represents a carbon with optionally one or more sharing        bonds;    -   R₆ represents nitrogen, oxygen, or sulfur or no atom (direct C—C        bond between aromatic ring and vinyl ring);    -   R₇ is optional and when present represents aromatic substitution        group to enhance the spectral properties such as increase        brightness and stability of the vinyl ether bridge;    -   R₈ is optional and when present represents linkers with aromatic        amino acids such as Phe, Trp, His or derivative thereof,        cationic amino acids such Arg, Lys, or derivative thereof,        anionic amino acids such as Asp, Glu or derivative of them,        unnatural amino acids of aromatic/cationic/anionic acids or        derivative thereof;    -   R₉ is optional and when present represents a linear carbon        chain, or polyethylene glycol linker, cationic linker, or        derivative thereof;    -   R₁₀ represents a CO₂H, PO₃H₂, SO₃H, CH₂SO₃H, CH₂CONHCH₂SO₃H,        CH₂CONHCH₂CH₂SO₃H;    -   R₁₁ represents CO₂H, SO₃H, CH₂CONHCH₂SO₃H, CH₂CONHCH₂CH₂SO₃H;        and    -   R₁₂ represents a hydrogen, a methyl group, a CH₂ and may        optionally represent each a CH₂ sharing a bond.

In some embodiments compounds of the present invention have anabsorption and emission maxima between about 500 nm and about 900 nm. Insome embodiments compounds of the present invention have an absorptionand emission maxima between about 600 nm and 800 nm.

In some embodiments compounds of the present invention are made tofluoresce after distribution thereof in the tissue cells. In someembodiments compounds of the present invention are made to fluoresce bysubjecting the compounds to excitation light of near infraredwavelength. In some embodiments compounds of the present invention havea binding affinity to PSMA that is similar to the binding affinity ofDUPA. In some embodiments compounds of the present invention are highlyselective for targeting to a tumor cell. In particularly preferredembodiments, the compounds of the present invention are targeted toprostate cancer cells.

In certain embodiments compounds of the present invention areadministered to a subject in need thereof and in some embodiments theadministered composition comprises, in addition to the compound, apharmaceutically acceptable carrier, excipient or diluent.

Some embodiments of the present invention provide methods of opticalimaging of PSMA-expressing biological tissue, said method comprising:

-   -   (a) contacting the biological tissue with a composition        comprising a PSMA-targeted NIR dye compound,    -   (b) allowing time for the compound in the composition to        distribute within the biological target;    -   (c) illuminating the tissue with an excitation light of a        wavelength absorbable by the compound; and    -   (d) detecting the optical signal emitted by the compound.

In some embodiments, these methods are used in detection of diseasesassociated with high PSMA expression. In some embodiments, furthercomprising the step of constructing an image from the signal emitted in(d). In some embodiments, the invention provides the aforementionedmethod wherein step (a) includes two or more fluorescent compounds whosesignal properties are distinguishable are contacted with the tissue, andoptionally the tissue is in a subject. In some embodiments the presentinvention provides use of an endoscope, catheter, tomographic system,hand-held optical imaging system, surgical goggles, or intra-operativemicroscope for the illuminating and/or detecting method steps.

In some embodiments, compositions and methods of the present inventionare used to treat cancer. In some embodiments, the cancer is selectedfrom the group consisting of prostate cancer, lung cancer, bladdercancer, pancreatic cancer, liver cancer, kidney cancer, sarcoma, breastcancer, brain cancer, neuroendocrine carcinoma, colon cancer, testicularcancer or melanoma. In some embodiments, PSMA-targeted NIR dye compoundsof the present invention are used for imaging of PSMA-expressing cells.In certain embodiments those cells are chosen from the group consistingof prostate cells, prostate cancer cells, bladder cancer cells,pancreatic cancer cells, liver cancer cells, lung cancer cells, kidneycancer cells, sarcoma cells, breast cancer cells, brain cancer cells,neuroendocrine carcinoma cells, colon cancer cells, testicular cancercells or melanoma cells.

The present invention also provides methods of targeting a cell type ina biological sample comprising: (a) contacting the biological samplewith a PSMA-targeted NIR dye compound for a time and under conditionsthat allow for binding of the compound to at least one cell of thetarget cell type; and (b) optically detecting the presence or absence ofthe compound of in the biological sample, wherein presence of thecompound in detecting step (b) indicates that the target cell type ispresent in the biological sample. In some embodiments the presentinvention provides methods for optical detection of PSMA-expressingcells comprising administering PSMA-targeting NIR dye compounds of thepresent invention and subjecting the compound to an excitation lightsource and detecting fluorescence from the compound. In someembodiments, the excitation light source is near-infrared wavelengthlight. In some embodiments the excitation light wavelength is within arange from about 600 to 1000 nanometers. In some embodiments theexcitation light wavelength is within a range from about 670 to 850nanometers.

In certain embodiments the present invention provides methods ofperforming image guided surgery on a subject comprising:

-   -   a) administering a composition comprising a PSMA-targeting NIR        dye compound under conditions and for a time sufficient for the        compound to accumulate at a given surgical site;    -   b) illuminating the compound to visualize the compound using        infrared light; and    -   c) performing surgical resection of the areas that fluoresce        upon excitation by the infrared light.

In some embodiments methods of the present invention the infrared lightwavelength is within a range from about 600 to 1000 nanometers. In someembodiments methods of the present invention use an infrared lightwavelength is within a range from about 670 to 850 nanometers.

Some embodiments of the present invention provide a method of diagnosinga disease in a subject comprising:

-   -   a) administering to a subject in need of diagnosis an amount of        a PSMA-targeted NIR dye compound for a time and under conditions        that allow for binding of the compound to at least one        PSMA-expressing cell;    -   b) measuring the signal from the compound of present in the        biological sample;    -   c) comparing the signal measured in b) with at least one control        data set, wherein the at least one control data set comprises        signals from the compound of claim 1 contacted with a biological        sample that does not comprise the target cell type; and    -   d) providing a diagnosis of disease wherein the comparison in        step c) indicates the presence of the disease.

Some embodiments of the present invention provide a kit comprising aPSMA-targeting NIR dye compound. In some embodiments, the kit is usedfor the imaging of PSMA-expressing cells. In some embodiments thePSMA-expressing cells are tumor cells. In some embodiments thePSMA-expressing cells are non-prostate cancer cells. In certainembodiments the PSMA-expressing cells are prostate tumor cells. Incertain embodiments the PSMA-expressing cells are cancer cells. Incertain embodiments the PSMA-expressing area is neo-vasculature of tumorcells. In some embodiments the present invention is used for detectionof metastatic disease. In some embodiments compounds of the presentinvention are used for improved surgical resection and/or improvedprognosis. In some embodiments methods of the present invention providecleaner surgical margins than non-NIR conjugated fluorescing dyes. Insome embodiments PSMA-targeted NIR dye compounds of the presentinvention have an improved tumor-to-background ratio.

In other embodiments compounds of the present invention are used toimage, diagnose, or detect non-prostate cancer cells chosen from thegroup consisting of bladder cancer cells, pancreatic cancer cells, livercancer cells, lung cancer cells, kidney cancer cells, sarcoma cells,breast cancer cells, brain cancer cells, neuroendocrine carcinoma cells,colon cancer cells, testicular cancer cells or melanoma cells. In otherembodiments, the cells being detected are more than 5 mm below the skin.In some embodiments, the tissue being detected is more than 5 mm belowthe skin. In other embodiments, the tumor being detected is more than 5mm below the skin. In some embodiments, the cells being detected aremore than 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm below the subject's skin. Insome embodiments of the present invention dye probes that are detectableoutside of the visible light spectrum. In some embodiments dye probesgreater than the visible light spectrum are used. In some embodimentscompounds of the present invention comprise dye probes sensitive towavelengths between 650 nm and 900 nm. In some embodiments thePSMA-targeted NIR dye compounds of the present invention have maximumlight absorption wavelengths in the near infrared region of betweenabout 650 nm and 1000 nm, for example and in one embodiment, atapproximately 800 nm.

In still another embodiment of the methods provided, the non-prostatecancer is bladder cancer, pancreatic cancer, liver cancer, lung cancer,kidney cancer, sarcoma, breast cancer, brain cancer, neuroendocrinecarcinoma, colon cancer, testicular cancer or melanoma.

In a further embodiment of the methods provided, the PSMA-expressingcancer cells are of a tumor. In still a further embodiment of themethods provided, the PSMA-expressing cancer is a tumor. In someembodiments, the volume of the tumor is at least 1000 mm³. In someembodiments, the volume of the tumor is less than 1000 mm³. In someembodiments, the volume of the tumor is less than 950 mm³. In someembodiments, the volume of the tumor is less than 900 mm³. In someembodiments, the volume of the tumor is less than 850 mm³. In someembodiments, the volume of the tumor is less than 800 mm³. In someembodiments, the volume of the tumor is less than 750 mm³. In someembodiments, the volume of the tumor is less than 700 mm³. In someembodiments, the volume of the tumor is less than 650 mm³. In someembodiments, the volume of the tumor is less than 600 mm³. In someembodiments, the volume of the tumor is less than 550 mm³. In someembodiments, the volume of the tumor is less than 500 mm³. In someembodiments, the volume of the tumor is less than 450 mm³. In someembodiments, the volume of the tumor is less than 400 mm³. In someembodiments, the volume of the tumor is less than 350 mm³. In someembodiments, the volume of the tumor is less than 300 mm³. In someembodiments, the volume of the tumor is less than 250 mm³. In someembodiments, the volume of the tumor is less than 200 mm³. In someembodiments, the volume of the tumor is less than 150 mm³. In someembodiments, the volume of the tumor is less than 100 mm³. In oneembodiment, the volume of the tumor is at least 75 mm³. In anotherembodiment, the volume of the tumor is less than 75 mm³. In anotherembodiment, the volume of the tumor is less than 70 mm³. In anotherembodiment, the volume of the tumor is less than 65 mm³. In anotherembodiment, the volume of the tumor is less than 60 mm³. In anotherembodiment, the volume of the tumor is less than 55 mm³. In oneembodiment, the volume of the tumor is at least 50 mm³. In otherembodiments, the tumor is less than 50 mm³. In another embodiment, thevolume of the tumor is less than 45 mm³. In other embodiments, thevolume of the tumor is less than 40 mm³. In another embodiment, thevolume of the tumor is less than 35 mm³. In still another embodiment,the volume of the tumor is less than 30 mm³. In another embodiment, thevolume of the tumor is less than 25 mm³. In still another embodiment,the volume of the tumor is less than 20 mm³. In another embodiment, thevolume of the tumor is less than 15 mm³. In still another embodiment,the volume of the tumor is less than 10 mm³. In still anotherembodiment, the volume of the tumor is less than 12 mm³. In stillanother embodiment, the volume of the tumor is less than 9 mm³. In stillanother embodiment, the volume of the tumor is less than 8 mm³. In stillanother embodiment, the volume of the tumor is less than 7 mm³. In stillanother embodiment, the volume of the tumor is less than 6 mm³. In stillanother embodiment, the volume of the tumor is less than 5 mm³.

In one embodiment, the tumor has a length of at least 5 mm prior tosurgical recession using a PSMA-targeted NIR dye compound of the presentinvention. In one embodiment, these methods detect tumors less than 5mm. In other embodiments the methods herein detect tumors less than 4mm. In some embodiments, the methods herein detect tumors less than 3mm. In another embodiment, the tumor has a length of at least 6 mm. Instill another embodiment, the tumor has a length of at least 7 mm. Inyet another embodiment, the tumor has a length of at least 8 mm. Inanother embodiment, the tumor has a length of at least 9 mm. In stillanother embodiment, the tumor has a length of at least 10 mm. In yetanother embodiment, the tumor has a length of at least 11 mm. In afurther embodiment, the tumor has a length of at least 12 mm. In still afurther embodiment, the tumor has a length of at least 13 mm. In still afurther embodiment, the tumor has a length of at least 14 mm. In anotherembodiment, the tumor has a length of at least 15 mm. In yet anotherembodiment, the tumor has a length of at least 16 mm. In still anotherembodiment, the tumor has a length of at least 17 mm. In a furtherembodiment, the tumor has a length of at least 18 mm. In yet a furtherembodiment, the tumor has a length of at least 19 mm. In still a furtherembodiment, the tumor has a length of at least 20 mm. In anotherembodiment, the tumor has a length of at least 21 mm. In still anotherembodiment, the tumor has a length of at least 22 mm. In yet anotherembodiment, the tumor has a length of at least 23 mm. In a furtherembodiment, the tumor has a length of at least 24 mm. In still a furtherembodiment, the tumor has a length of at least 25 mm. In yet a furtherembodiment, the tumor has a length of at least 30 mm.

In some embodiments the present disclosure relates to prostate specificmembrane antigen (PSMA) targeted compounds conjugated to near-infra red(NIR) dyes and methods for their therapeutic and diagnostic use. Morespecifically, this disclosure provides compounds and methods fordiagnosing and treating diseases associated with cells expressingprostate specific membrane antigen (PSMA), such as prostate cancer,solid tumors, and related diseases. The disclosure further describesmethods and compositions for making and using the compounds, methodsincorporating the compounds, and kits incorporating the compounds. Ithas been discovered that a PSMA-targeted compound, such as DUPAconjugated to an NIR dye via a linker (L) may be useful in the imaging,diagnosis, and/or treatment of prostate cancer, and related diseasesthat involve pathogenic cell populations expressing or over-expressingPSMA. PSMA is a cell surface protein that is internalized in a processanalogous to endocytosis observed with cell surface receptors, such asvitamin receptors. Accordingly, it has been discovered that certainconjugates that include a linker having a predetermined length, and/or apredetermined diameter, and/or preselected functional groups along itslength may be used to treat, image, and/or diagnose such diseases.

In one illustrative embodiment, the linker L may be a releasable ornon-releasable linker. In one aspect, the linker L is at least about 7atoms in length. In one variation, the linker L is at least about 10atoms in length. In one variation, the linker L is at least about 14atoms in length. In another variation, the linker L is between about 7and about 22, between about 7 and about 20, or between about 7 and about18 atoms in length. In another variation, the linker L is between about14 and about 22, between about 15 and about 12, or between about 14 andabout 20 atoms in length.

In an alternative aspect, the linker L is at least about 10 angstroms(Å) in length.

In one variation, the linker L is at least about 15 Å in length. Inanother variation, the linker L is at least about 20 Å in length. Inanother variation, the linker L is in the range from about 10 Å to about30 Å in length.

In an alternative aspect, at least a portion of the length of the linkerL is about 5 Å in diameter or less at the end connected to the bindingligand B. In one variation, at least a portion of the length of thelinker L is about 4 Å or less, or about 3 Å or less in diameter at theend connected to the binding ligand B. It is appreciated that theillustrative embodiments that include a diameter requirement of about 5Å or less, about 4 Å or less, or about 3 Å or less may include thatrequirement for a predetermined length of the linker, thereby defining acylindrical-like portion of the linker. Illustratively, in anothervariation, the linker includes a cylindrical portion at the endconnected to the binding ligand that is at least about 7 Å in length andabout 5 Å or less, about 4 Å or less, or about 3 Å or less in diameter.

In another embodiment, the linker L includes one or more hydrophiliclinkers capable of interacting with one or more residues of PSMA,including amino acids that have hydrophilic side chains, such as Ser,Thr, Cys, Arg, Orn, Lys, Asp, Glu, Gln and like residues. In anotherembodiment, the linker L includes one or more hydrophobic linkerscapable of interacting with one or more residues of PSMA, includingamino acids that have hydrophobic side chains, such as Val, Leu, Phe,Tyr, Met, and like residues. It is to be understood that the foregoingembodiments and aspects may be included in the linker L either alone orin combination with each other. For example, linkers L that are at leastabout 7 atoms in length and about 5 Å, about 4 Å or less, or about 3 Åor less in diameter or less are contemplated and described herein, andalso include one or more hydrophilic linkers capable of interacting withone or more residues of PSMA, including Val, Leu, Phe, Tyr, Met, andlike residues are contemplated and described herein.

In another embodiment, one end of the linker is not branched andcomprises a chain of carbon, oxygen, nitrogen, and sulfur atoms. In oneembodiment, the linear chain of carbon, oxygen, nitrogen, and sulfuratoms is at least 5 atoms in length. In one variation, the linear chainis at least 7 atoms, or at least 10 atoms in length. In anotherembodiment, the chain of carbon, oxygen, nitrogen, and sulfur atoms arenot substituted. In one variation, a portion of the chain of carbon,oxygen, nitrogen, and sulfur atoms is cyclized with a divalent fragment.For example, a linker (L) comprising the dipeptide Phe-Phe may include apiperazin-1,4-diyl structure by cyclizing two nitrogens with an ethylenefragment, or substituted variation thereof.

In another embodiment, pharmaceutical compositions are described herein,where the pharmaceutical composition includes the conjugates describedherein in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA. Illustratively, the pharmaceutical compositionsalso include one or more carriers, diluents, and/or excipients.

In another embodiment, methods for treating diseases and disease states,diagnosing diseases or disease states, and/or imaging tissues and/orcells that are associated with pathogenic populations of cellsexpressing or over expressing PSMA are described herein. Such methodsinclude the step of administering the conjugates described herein,and/or pharmaceutical compositions containing the conjugates describedherein, in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1—Synthesis of DUPA-Linker-NIR dye conjugates

FIG. 2A—Structure of PSMA-targeted DUPA-FITC (Fluoresceinisothiocyanate) conjugate (14).

FIG. 2(B)—PSMA-targeted DUPA-FITC (Fluorescein isothiocyanate) conjugate(14) and its binding affinity (K_(D)) and specificity on PSMA-positive22Rv1 human prostate cancer cells and on PSMA-negative A549 humanalveolar basal epithelial cells in culture. DUPA-FITC dissolved in RPMImedium was added at the indicated concentrations to 22Rv1 or A549 cellsin RPMI culture media and allowed to incubate for 1 h at 37° C. Mediawas then removed, washed with fresh media (3x), and replaced with PBS(phosphate buffered saline). Samples were analyzed using flow cytometry.Error bars represent SD (n=3). s are contained within the antigenrecognition site.

FIG. 3—Relative binding affinities of DUPA-NIR conjugates 1-9 withrespect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancercells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry.

FIG. 4A—Tumor-to-tissue fluorescence ratio from tissue biodistributiondata of PSMA-targeted DUPA-NIR conjugates conjugates 1-9

FIG. 4B—After imaging the tumor-to-tissue fluorescence ratio from tissuebiodistribution data of PSMA-targeted DUPA-NIR conjugates, fluorescencewithin a region of interest (ROI) was measured for each tissue using InVivo imaging software and tumor-to-tissue fluorescence was thencalculated.

FIG. 5—Structures of PSMA-targeted DUPA-Linker-NIR imaging agents witharomatic amino acid linkers between the ligand and the NIR dye

FIG. 6—Relative binding affinities of DUPA-NIR conjugates with aromaticamino acids linkers with respect to DUPA-FITC (14). PSMA-positive 22Rv1human prostate cancer cells were incubated for 1 h at 37° C. in thepresence of 100 nM DUPA-FITC with increasing concentrations of DUPA-NIRconjugates. Media was then removed, washed with fresh media (3x), andreplaced with PBS. Cell bound fluorescence was assayed as using flowcytometry.

FIG. 7A—Tissue biodistribution analysis of DUPA-NIR conjugates 15 and 23using fluorescence imaging of mice bearing human prostate tumorxenografts (22Rv1 cells). Male nude mice with 22Rv1 tumor xenograftswere injected with DUPA-NIR dye conjugates via tail vein. The mice wereeuthanized 2 h after administration of the DUPA-NIR dye conjugate,selected tissues were harvested, and tissues were imaged with IVISimager (ex=745 nm, em=ICG, exposure time=1 s). After imaging,fluorescence within a Region of interest (ROI) was measured for eachtissue using In Vivo imaging software and tumor-to-tissue fluorescencewas then calculated.

FIG. 7B—Tumor-to-tissue ratio of DUPA-NIR conjugates 15 and 23 usingfluorescence imaging of mice bearing human prostate tumor xenografts(22Rv1 cells). Male nude mice with 22Rv1 tumor xenografts were injectedwith DUPA-NIR dye conjugates via tail vein. The mice were euthanized 2 hafter administration of the DUPA-NIR dye conjugate, selected tissueswere harvested, and tissues were imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s). After imaging, fluorescence within a Regionof interest (ROI) was measured for each tissue using In Vivo imagingsoftware and tumor-to-tissue fluorescence was then calculated.

FIG. 8—Overlay of whole or half body fluorescence image over white lightimages after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 14 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 9—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 23 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 10—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 25 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 11—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 6 nmol of 35 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 12—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 6 nmol of 36 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 13—Structures of PSMA-targeted DUPA-Linker-NIR imaging agents withpositive charge linkers between the ligand and the NIR dye

FIG. 14—Relative binding affinities of DUPA-NIR conjugates with respectto DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancer cells wereincubated for 1 h at 37° C. in the presence of 100 nM DUPA-FITC withincreasing concentrations of DUPA-NIR conjugates. Media was thenremoved, washed with fresh media (3x), and replaced with PBS. Cell boundfluorescence was assayed as using flow cytometry.

FIG. 15—Tumor-to-tissue ratio of DUPA-NIR conjugates 39 and 41 usingfluorescence imaging of mice bearing human prostate tumor xenografts (22Rv1 cells). Male nude mice with 22Rv1 tumor xenografts were injectedwith DUPA-NIR dye conjugates via tail vein. The mice were euthanized 2 hafter administration of the DUPA-NIR dye conjugate, selected tissueswere harvested, and tissues were imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s). After imaging, fluorescence within a Regionof interest (ROI) was measured for each tissue using In Vivo imagingsoftware and tumor-to-tissue fluorescence was then calculated.

FIG. 16—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 39 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 17—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 40 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 18—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 41 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 19—Structures of PSMA-targeted DUPA-Linker-NIR imaging agents withnegative charge linkers between the ligand and the NIR dye

FIG. 20—Relative binding affinities of DUPA-NIR conjugates of 49 and 50with respect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostatecancer cells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry.

FIG. 21A—Tissue biodistribution analysis of DUPA-NIR conjugates 49 and50 using fluorescence imaging of mice bearing human prostate tumorxenografts (22Rv1 cells). Male nude mice with 22Rv1 tumor xenograftswere injected with DUPA-NIR dye conjugates via tail vein. The mice wereeuthanized 2 h after administration of the DUPA-NIR dye conjugate,selected tissues were harvested, and tissues were imaged with IVISimager (ex=745 nm, em=ICG, exposure time=1 s). After imaging,fluorescence within a Region of interest (ROI) was measured for eachtissue using In Vivo imaging software and tumor-to-tissue fluorescencewas then calculated.

FIG. 21B—Tumor-to-tissue ratio of DUPA-NIR conjugates 49 and 50 usingfluorescence imaging of mice bearing human prostate tumor xenografts(22Rv1 cells). Male nude mice with 22Rv1 tumor xenografts were injectedwith DUPA-NIR dye conjugates via tail vein. The mice were euthanized 2 hafter administration of the DUPA-NIR dye conjugate, selected tissueswere harvested, and tissues were imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s). After imaging, fluorescence within a Regionof interest (ROI) was measured for each tissue using In Vivo imagingsoftware and tumor-to-tissue fluorescence was then calculated.

FIG. 22—Structures of PSMA-targeted DUPA-Linker-NIR imaging agents withvariably charged NIR dye molecule

FIG. 23—Relative binding affinities of DUPA-NIR conjugates with respectto DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancer cells wereincubated for 1 h at 37° C. in the presence of 100 nM DUPA-FITC withincreasing concentrations of DUPA-NIR conjugates. Media was thenremoved, washed with fresh media (3x), and replaced with PBS. Cell boundfluorescence was assayed as using flow cytometry.

FIG. 24—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 54 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 25—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 55 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 26—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 56 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 27—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 57 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 28—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 58 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 29—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 60 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 30—Structures of PSMA-targeted DUPA-Linker-NIR imaging agents withmiscellaneous linkers and NIR dyes

FIG. 31—Relative binding affinities of DUPA-NIR conjugates with respectto DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancer cells wereincubated for 1 h at 37° C. in the presence of 100 nM DUPA-FITC withincreasing concentrations of DUPA-NIR conjugates. Media was thenremoved, washed with fresh media (3x), and replaced with PBS. Cell boundfluorescence was assayed as using flow cytometry.

FIG. 32—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 63 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 33—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 6 nmol of 63 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals.

FIG. 34—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 20 nmol of 64 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at different timeintervals

FIG. 35—Structures of PSMA-targeted NIR imaging agents with differentligands

FIG. 36—Relative binding affinities of PSMA-targeted NIR conjugates withrespect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancercells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry.

FIG. 37—Overlay of whole body or half body fluorescence image over whitelight images after adjusting the threshold. 22Rv1 human prostate tumorxenograft bearing mouse was injected with 6 nmol of 14 and imaged withIVIS imager (ex=745 nm, em=ICG, exposure time=1s) at different timeintervals.

DEFINITIONS

It is to be understood that this invention is not limited to theparticular methodology, protocols, cell lines, constructs, and reagentsdescribed herein and as such may vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention, which will be limited only by the appended claims.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. Thus, for example, reference to a “prostate specific membraneantigen ligand” “PSMA ligand” is a reference to one or more such ligandsand includes equivalents thereof known to those skilled in the art, andso forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devices,and materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications and patents mentioned herein are incorporated herein byreference for the purpose of describing and disclosing, for example, theconstructs and methodologies that are described in the publications,which might be used in connection with the presently describedinvention. The publications discussed herein are provided solely fortheir disclosure prior to the filing date of the present application.Nothing herein is to be construed as an admission that the inventors arenot entitled to antedate such disclosure by virtue of prior invention orfor any other reason.

With respect to PSMA-targeted NIR conjugates of the present invention,the term “antigenically specific” or “specifically binds” refers toPSMA-targeting compounds that bind to one or more epitopes of PSMA, butwhich do not substantially recognize and bind other molecules in asample containing a mixed population of antigens.

The term “epitope” as used herein refers to a site on PSMA that isrecognized by DUPA. An epitope may be a linear or conformationallyformed sequence or the shape of amino acids.

As used herein, “PSMA-targeting compound” or “PSMA-targeted compound”shall include those small molecules, ligands, polypeptides and proteinsthat have at least the biological activity of specific binding to PSMAor an epitope of PSMA. These compounds include ligands, receptors,peptides, or any amino acid sequence that binds to PSMA or to at leastone PSMA epitope.

Compounds of the present invention comprise a PSMA-targeting compound,they may bind a portion of PSMA itself, or they may bind a cell surfaceprotein or receptor that is associated with PSMA.

The terms “functional group”, “active moiety”, “activating group”,“leaving group”, “reactive site”, “chemically reactive group” and“chemically reactive moiety” are used in the art and herein to refer todistinct, definable portions or units of a molecule. The terms aresomewhat synonymous in the chemical arts and are used herein to indicatethe portions of molecules that perform some function or activity and arereactive with other molecules.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acid analogs and amino acidmimetics that function in a manner similar to the naturally occurringamino acids. Naturally encoded amino acids are the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and pyrolysine and selenocysteine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, such as,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (such as, norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission.

The present invention addresses, among other things, problems associatedwith the early diagnosis and surgical treatment of PSMA-expressing cellsinvolved in disease and/or cancer, and in particular PSMA-targeted dyeconjugates with improved imaging, diagnostic, biological propertiesincluding, as non-limiting examples, higher specificity, decreasedbackground signal and increased tumor fluorescence.

DETAILED DESCRIPTION

Surgery cures 50% of patients with solid tumors in the US, while chemo-and radiotherapy cure less than 5% of all cancer patients. Over 700,000patients undergo cancer surgery every year in the US and 40% of surgicalpatients have a recurrence of locoregional disease within 5 years.Despite major advances in the field of oncology there remains a need forearly detection, methods to overcome hurdles to complete surgicalresection of the primary tumor with negative margins, and removal ofmetastatic cancer cells and identification of satellite disease.Achieving these three goals not only improves disease clearance but alsoguides decisions regarding postoperative chemotherapy and radiation.While non-targeted fluorescent dyes have been shown to passivelyaccumulate in some tumors, the resulting tumor-to-background ratios areoften poor and the boundaries between malignant and healthy tissues canbe difficult to define. Although ligand targeted fluorescence dyes(e.g., EC17: Folate-EDA-FITC) have been used for imaging a tissue, thosedyes have been ineffective as they would not penetrate deep tissue andhence only identified the specific cells on the surface of a tissuerather than deeper within the tissue sample. In addition,fluorescein-based dyes have the disadvantages that of low shelf-lifestability. Thiourea bridge formed by Fluorescence isothiocynate (FITC)compounds easily decomposes making unstable compound. In addition, asEC17 uses fluorescein which has the drawback of a relatively high levelof nonspecific background noise from collagen in the tissues surroundingthe imaging site. Moreover, the absorption of visible light bybiological chromophores, in particular hemoglobin, further limits theusefulness of dyes that incorporate fluorescein. Therefore, conventionaldyes cannot readily detect tumors that may be buried deeper than a fewmillimeters in the tissue. Furthermore, fluorescence from fluorescein isquenched at low pH (below pH 5).

In order for a dye material to be useful in detecting and guidingsurgery or providing detection of early, metastatic, and other tissueimaging it is important to overcome these drawbacks. The presentinvention provides PSMA-targeted conjugates of near infrared dyes thatare stable, fluoresce in the infrared range, penetrate deep withintargeted tissue to produce a specific and bright identification of areasof tissue that express PSMA, fast clearance from tissues that do notexpress PSMA to obtain high tumor-to-background ratio, and fast skinclearance. More specifically, the PSMA-targeted conjugates are linked tothe near infrared dyes through a linker consisting of one or more atomicspacers, amino acids, amino acid derivatives. Even more specifically, ithas been found that where the atomic spacer is hydrophobic 7-atom spacerwith neutral or charged atoms and amino acid spacer is aromatic aminoacid or a derivative of aromatic amino acid, or negative or positivecharge amino acid and tyrosine or a derivative of tyrosine. Charge ofthe linker can be varied to obtain fast skin clearance and fast tumoraccumulation to obtain higher tumor-to-background ratio. Moreover, thefluorescence intensity of the NIR dye is maintained or even enhanced byhaving the aromatic amino acid or tyrosine or derivative of tyrosine andcharge of the NIR dye can be varied to accomplish fast skin clearance.

This disclosure provides PSMA-targeted ligands linked to NIR dyes andmethods for synthesizing the same. This disclosure also providescompounds for use in the targeted imaging of tumors expressing PSMA,including but not limited to prostate cancer, and methods of use, forexample, in imaging and surgery involving PSMA positive tissues andtumors.

In certain embodiments, compounds of the present invention have theform: B-X-Y-Z

-   -   wherein B is a PSMA-targeted compound;    -   X is a spacer;    -   Y is an amino acid spacer; and    -   Z is an NIR dye.

In some embodiments, the PSMA-targeted compound is chosen from the groupconsisting of a small molecule, a ligand, or a derivative thereof. Insome embodiments, the PSMA-targeted compound is a ligand. In someembodiments, the PSMA-targeted compound is DUPA. In other embodiments,the PSMA-targeted compound is a small molecule that binds PSMA.

In some embodiments, X is a hydrophobic spacer. In some embodoiments, Xis selected from the group consisting of an eight aminooctonoic acid(EAOA), a chain of 7 atoms, polyethylene glycol spacer, a spacer 7 atomsin length, cationic spacer, chain of 7 atoms, a chain from 7 to 24 atomsin length; a peptide comprising two aryl or aryl alkyl groups, each ofwhich is optionally substituted, and where one aryl or aryl alkyl groupis about 7 to about 11, or about 7 to about 14 atoms, and the other arylor aryl alkyl group is about 10 to about 14, or about 10 to about 17atoms. In another embodiment, the spacer ccomprises about 1 to about 30atoms, or about 2 to about 20 atoms. In some embodiments, the spacer is7 atoms in length. In some embodiments, the spacer comprises EAOA. Insome embodiments, the spacer is variably charged. In some embodiments, Xhas a positive charge. In other embodiments, X has a negative charge.

In some embodiments, Y is selected from the group consisting of: acidic(negatively charged) amino acids, such as aspartic acid and glutamicacid; basic (positively charged) amino acids such as arginine,histidine, and lysine; neutral polar amino acids, such as glycine,serine, threonine, cysteine, tyrosine, asparagine, and glutamine;neutral nonpolar (hydrophobic) amino acids, such as alanine, leucine,isoleucine, valine, proline, phenylalanine, tryptophan, and methionine;and derivatives thereof. In some embodiments, Y is an aromatic aminoacid. In some embodiments, Y has a positive charge. In otherembodiments, Y has a negative charge.

In some embodiments, Z is selected from the group consisting ofnear-infra red dyes, including but not limited to, LS288, IR800, SP054,S0121, KODAK, S2076 S0456 and/or the dyes selected from group consistingof.

In certain embodiments, the Z is variably charged. In some embodiments,Z has a positive charge. In other embodiments, Z has a negative charge.

In certain embodiments, compounds of the present invention have theform:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Y is an amino acidspacer with a sulfur-containing side chain group; and Z is an NIR dye.In some embodiments, the amino acid spacer with a sulfur-containing sidegroup is cysteine. In some embodiments, the amino acid spacer with asulfur-containing side group is methionine. In some embodiments, theamino acid spacer with a sulfur-containing side group is moleculecontaining thiophenol moiety. In some embodiments, compounds of thepresent invention have the form:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Y is an amino acidspacer with a chalcogen-containing side chain group; and Z is an NIRdye. In some embodiments the present invention provides compounds of theform:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Y is an amino acidchosen from the group consisting of tyrosine, cysteine, lysine, or aderivative thereof; and Z is an NIR dye. In some embodiments, Ycomprises a tyrosine or tyrosine derivative. In some embodiments, Ycomprises a tyrosine and a carbon isotope is on the aromatic ring oftyrosine. In some embodiments, Y comprises an amino acid with anaromatic ring with a hydrogen isotope.

In some embodiments, compounds of the present invention have the form:B-X-Y-Z

wherein B is a PSMA-targeted compound; X is a spacer; Z is an NIR dye;and Y comprises a derivative of tyrosine selected from the groupconsisting of:

or racemic mixtures thereof.

In some embodiments the invention includes the compound B-X-Y-Z whereinB comprises DUPA or a derivative thereof, X comprises an EAOA, Ycomprises tyrosine, and Z comprises S0456.

A compound having the structural formula:

-   -   or a pharmaceutically acceptable salt thereof, or isotopes        thereof, wherein:    -   R₁ represents a hydrogen or SO₃H;    -   R₂ represents a hydrogen, CH₃, C₃H₆SO₃ ⁻, C₃H₆SO₃H or C₄H₈SO₃ ⁻,        or C₄H₈SO₃H or C₃H₆N⁺(CH₃)₃;    -   R₃, and R₅ each represents a carbon, optionally one or more        sharing bonds,    -   R₄ represents a carbon with optionally one or more sharing        bonds;    -   R₆ represents nitrogen, oxygen, or sulfur or no atom (direct C—C        bond between aromatic ring and vinyl ring);    -   R₇ is optional and when present represents aromatic substitution        group to enhance the spectral properties such as increase        brightness and stability of the vinyl ether bridge;    -   R₈ is optional and when present represents linkers with aromatic        amino acids such as Phe, trp, His or derivative of them,        cationic amino acids such Arg, Lys, or derivative of them,        anionic amino acids such as Asp, Glu or derivative of them,        unnatural amino acids of aromatic/cationic/anionic acids or        derivative;    -   R₉ is optional and when present represents a linear carbon        chain, or polyethylene glycol linker, cationic linker, or        derivative of them;    -   R₁₀ represents a CO₂H, PO₃H₂, SO₃H, CH₂SO₃H, CH₂CONHCH₂SO₃H,        CH₂CONHCH₂CH₂S 0₃H;    -   R₁₁ represents CO₂H, SO₃H, CH₂CONHCH₂SO₃H, CH₂CONHCH₂CH₂SO₃H;        and    -   R₁₂ represents a hydrogen, a methyl group, a CH₂ and may        optionally represent each a CH₂ sharing a bond.

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

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In some embodiments the present invention includes a compound that hasthe structural formula:

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In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention a compound that has thestructural formula:

In some embodiments the present invention a compound that has thestructural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

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In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

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In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

In some embodiments the present invention includes a compound that hasthe structural formula:

Additional preferred compounds of the invention include the following:

and

A compound of structure 35 is particularly preferred in the presentinvention.

In addition, stereoisomers of compound 35 such as those shown in thefollowing table also are contemplated to be useful PSMA-targetednear-infra red (NIR) dyes for use in the methods of the presentinvention.

Chiral Center Compound 1* 2* 3* 4*  35 L L L L 114 L L L D 115 L L D L116 L L D D 117 L D L L 118 L D L D 119 L D D L 120 L D D D 121 D L L L122 D L L D 123 D L D L 124 D L D D 125 D D L L 126 D D L D 127 D D D L128 D D D D Note: Chiral center is indicated as *

Additional preferred compounds of the invention include the following:

-   -   or a pharmaceutically acceptable salt thereof, or isotopes        thereof, wherein:    -   R₁ represents a hydrogen or SO₃H;    -   R₂ represents a hydrogen, or CH₃, or C₃H₆SO₃ ⁻, or C₃H₆SO₃H or        C₄H₈SO₃ ⁻, or C₄H₈SO₃H or C₃H₆N⁺(CH₃)₃;    -   R₃, and R₅ each represents a carbon, optionally one or more        sharing bonds, or oxygen, or sulfur, or nitrogen    -   R₄ represents a carbon with optionally one or more sharing        bonds;    -   R₆ represents nitrogen, oxygen, or sulfur or no atom (direct C—C        bond between aromatic ring and vinyl ring);    -   R₇ is optional and when present represents electron donating        aromatic substitution group;    -   R₈ is optional and when present represents linkers with aromatic        amino acids such as Phe, Trp, His, Tyr, or derivative of them,        and/or cationic amino acids such Arg, Lys, or derivative of        them, and/or anionic amino acids such as Asp, Glu or derivative        of them, and/or unnatural amino acids of        aromatic/cationic/anionic acids or derivative;    -   R₉ is optional and when present represents a linear carbon        chain, or polyethylene glycol linkers, polyethylene amine        linkers, cationic linker, or derivative of them;    -   R₁₀ represents a CO₂H, PO₃H₂, SO₃H, CH₂SO₃H, CH₂CONHCH₂SO₃H,        CH₂CONHCH₂CH₂SO₃H;    -   R₁₁ represents CO₂H, SO₃H, CH₂CONHCH₂SO₃H, CH₂CONHCH₂CH₂SO₃H;        and    -   R₁₂ represents independently represents a hydrogen, a methyl        group, CH₂COOH, a CH₂ and may optionally represent each a CH₂        sharing a bond.

Additional preferred compounds of the invention include the following:

Additional preferred compounds of the invention include the following:

-   -   or a pharmaceutically acceptable salt thereof, or isotopes        thereof, wherein:    -   R₁ represents a hydrogen or SO₃H;    -   R₂ represents a hydrogen, or CH₃, or C₃H₆SO₃ ⁻, or C₃H₆SO₃H or        C₄H₈SO₃ ⁻, or C₄H₈SO₃H or C₃H₆N⁺(CH₃)₃;    -   R₃, and R₅ each represents a carbon, optionally one or more        sharing bonds, or oxygen, or sulfur, or nitrogen    -   R₄ represents a carbon with optionally one or more sharing        bonds;    -   R₆ represents nitrogen, oxygen, or sulfur or no atom (direct C—C        bond between aromatic ring and vinyl ring);    -   R₇ is optional and when present represents electron donating        aromatic substitution group;    -   R₈ is optional and when present represents linkers with aromatic        amino acids such as Phe, Trp, His, Tyr, or derivative of them,        and/or cationic amino acids such Arg, Lys, or derivative of        them, and/or anionic amino acids such as Asp, Glu or derivative        of them, and/or unnatural amino acids of        aromatic/cationic/anionic acids or derivative;    -   R₉ is optional and when present represents a linear carbon        chain, or polyethylene glycol linkers, polyethylene amine        linkers, cationic linker, or derivative of them;    -   R₁₀ represents a CO₂H, PO₃H₂, SO₃H, CH₂SO₃H, CH₂CONHCH₂SO₃H,        CH₂CONHCH₂CH₂SO₃H;    -   R₁₁ represents CO₂H, SO₃H, CH₂CONHCH₂SO₃H, CH₂CONHCH₂CH₂SO₃H;        and    -   R₁₂ represents independently represents a hydrogen, a methyl        group, CH₂COOH, a CH₂ and may optionally represent each a CH₂        sharing a bond.

Additional preferred compounds of the invention include the following:

In some embodiments compounds of the present invention have anabsorption and emission maxima between about 500 nm and about 900 nm. Insome embodiments compounds of the present invention have an absorptionand emission maxima between about 600 nm and 800 nm.

In some embodiments compounds of the present invention are made tofluoresce after distribution thereof in the tissue cells. In someembodiments compounds of the present invention are made to fluoresce bysubjecting the compounds to excitation light of near infraredwavelength. In some embodiments compounds of the present invention havea binding affinity to PSMA that is similar to the binding affinity ofDUPA. In some embodiments compounds of the present invention are highlyselective for targeting to a tumor cell.

In certain embodiments compounds of the present invention areadministered to a subject in need thereof and in some embodiments theadministered composition comprises, in addition to the compound, apharmaceutically acceptable carrier, excipient or diluent.

Some embodiments of the present invention provide methods of opticalimaging of PSMA-expressing biological tissue, said method comprising:

-   -   (a) contacting the biological tissue with a composition        comprising a PSMA-targeted NIR dye compound,    -   (b) allowing time for the compound in the composition to        distribute within the biological target;    -   (c) illuminating the tissue with an excitation light of a        wavelength absorbable by the compound; and    -   (d) detecting the optical signal emitted by the compound.

In some embodiments, these methods are used in detection of diseasesassociated with high PSMA expression. In some embodiments, furthercomprising the step of constructing an image from the signal emitted in(d). In some embodiments, the invention provides the aforementionedmethod wherein step (a) includes two or more fluorescent compounds whosesignal properties are distinguishable are contacted with the tissue, andoptionally the tissue is in a subject. In some embodiments the presentinvention provides use of an endoscope, catheter, tomographic system,hand-held optical imaging system, surgical goggles, or intra-operativemicroscope for the illuminating and/or detecting method steps.

In some embodiments, compositions and methods of the present inventionare used to treat cancer. In some embodiments, the cancer is selectedfrom the group consisting of prostate cancer, bladder cancer, pancreaticcancer, liver cancer, lung cancer, kidney cancer, sarcoma, breastcancer, brain cancer, neuroendocrine carcinoma, colon cancer, testicularcancer or melanoma. In some embodiments, PSMA-targeted NIR dye compoundsof the present invention are used for imaging of PSMA-expressing cells.In certain embodiments those cells are chosen from the group consistingof prostate cells, prostate cancer cells, bladder cancer cells,pancreatic cancer cells, liver cancer cells, lung cancer cells, kidneycancer cells, sarcoma cells, breast cancer cells, brain cancer cells,neuroendocrine carcinoma cells, colon cancer cells, testicular cancercells or melanoma cells;

The present invention also provides methods of targeting a cell type ina biological sample comprising: a) contacting the biological sample witha PSMA-targeted NIR dye compound for a time and under conditions thatallow for binding of the compound to at least one cell of the targetcell type; and b) optically detecting the presence or absence of thecompound of in the biological sample, wherein presence of the compoundin detecting step c) indicates that the target cell type is present inthe biological sample. In some embodiments the present inventionprovides methods for optical detection of PSMA-expressing cellscomprising administering PSMA-targeting NIR dye compounds of the presentinvention and subjecting the compound to an excitation light source anddetecting fluorescence from the compound. In some embodiments, theexcitation light source is near-infrared wavelength light. In someembodiments the excitation light wavelength is within a range from about600 to 1000 nanometers. In some embodiments the excitation lightwavelength is within a range from about 670 to 850 nanometers.

In certain embodiments the present invention provides methods ofperforming image guided surgery on a subject comprising:

-   -   a) administering a composition comprising a PSMA-targeting NIR        dye compound under conditions and for a time sufficient for the        compound to accumulate at a given surgical site;    -   b) illuminating the compound to visualize the compound using        infrared light; and    -   c) performing surgical resection of the areas that fluoresce        upon excitation by the infrared light.

In some embodiments methods of the present invention the infrared lightwavelength is within a range from about 600 to 1000 nanometers. In someembodiments methods of the present invention use an infrared lightwavelength is within a range from about 670 to 850 nanometers.

Some embodiments of the present invention provide a method of diagnosinga disease in a subject comprising:

-   -   a) administering to a subject in need of diagnosis an amount of        a PSMA-targeted NIR dye compound for a time and under conditions        that allow for binding of the compound to at least one        PSMA-expressing cell or tissues (PSMA also express in        neo-vasculature of most of the solid tumors);    -   b) measuring the signal from the compound of present in the        biological sample;    -   c) comparing the signal measured in b) with at least one control        data set, wherein the at least one control data set comprises        signals from the compound of claim 1 contacted with a biological        sample that does not comprise the target cell type; and    -   d) providing a diagnosis of disease wherein the comparison in        step c) indicates the presence of the disease.

Some embodiments of the present invention provide a kit comprising aPSMA-targeting NIR dye compound. In some embodiments, the kit is usedfor the imaging of PSMA-expressing cells or tissues. In some embodimentsthe PSMA-expressing cells are tumor cells. In some embodiments thePSMA-expressing cells are non-prostate cancer cells. In certainembodiments the PSMA-expressing cells are prostate tumor cells. Incertain embodiments the PSMA-expressing cells are cancer cells. In someembodiments the present invention is used for detection of metastaticdisease. In some embodiments compounds of the present invention are usedfor improved surgical resection and/or improved prognosis. In someembodiments methods of the present invention provide cleaner surgicalmargins than non-NIR conjugated fluorescing dyes. In some embodimentsPSMA-targeted NIR dye compounds of the present invention have animproved tumor-to-background ratio.

In other embodiments compounds of the present invention are used toimage, diagnose, or detect non-prostate cancer cells chosen from thegroup consisting of bladder cancer cells, pancreatic cancer cells, livercancer cells, lung cancer cells, kidney cancer cells, sarcoma cells,breast cancer cells, brain cancer cells, neuroendocrine carcinoma cells,colon cancer cells, testicular cancer cells or melanoma cells. In otherembodiments, the cells being detected are more than 5 mm below the skin.In some embodiments, the tissue being detected is more than 5 mm belowthe skin. In other embodiments, the tumor being detected is more than 5mm below the skin. In some embodiments, the cells being detected aremore than 6 mm, 7 mm, 8 mm, 9 mm, or 10 mm below the subject's skin. Insome embodiments of the present invention dye probes that are detectableoutside of the visible light spectrum. In some embodiments dye probesgreater than the visible light spectrum are used. In some embodimentscompounds of the present invention comprise dye probes sensitive towavelengths between 650 nm and 900 nm. In some embodiments thePSMA-targeted NIR dye compounds of the present invention have maximumlight absorption wavelengths in the near infrared region of betweenabout 650 nm and 1000 nm, for example and in one embodiment, atapproximately 800 nm.

In still another embodiment of the methods provided, the non-prostatecancer is bladder cancer, pancreatic cancer, liver cancer, lung cancer,kidney cancer, sarcoma, breast cancer, brain cancer, neuroendocrinecarcinoma, colon cancer, testicular cancer or melanoma.

In a further embodiment of the methods provided, the PSMA-expressingcancer cells are of a tumor. In still a further embodiment of themethods provided, the PSMA-expressing cancer is a tumor. In someembodiments, the volume of the tumor is at least 1000 mm³. In someembodiments, the volume of the tumor is less than 1000 mm³. In someembodiments, the volume of the tumor is less than 950 mm³. In someembodiments, the volume of the tumor is less than 900 mm³. In someembodiments, the volume of the tumor is less than 850 mm³. In someembodiments, the volume of the tumor is less than 800 mm³. In someembodiments, the volume of the tumor is less than 750 mm³. In someembodiments, the volume of the tumor is less than 700 mm³. In someembodiments, the volume of the tumor is less than 650 mm³. In someembodiments, the volume of the tumor is less than 600 mm³. In someembodiments, the volume of the tumor is less than 550 mm³. In someembodiments, the volume of the tumor is less than 500 mm³. In someembodiments, the volume of the tumor is less than 450 mm³. In someembodiments, the volume of the tumor is less than 400 mm³. In someembodiments, the volume of the tumor is less than 350 mm³. In someembodiments, the volume of the tumor is less than 300 mm³. In someembodiments, the volume of the tumor is less than 250 mm³. In someembodiments, the volume of the tumor is less than 200 mm³. In someembodiments, the volume of the tumor is less than 150 mm³. In someembodiments, the volume of the tumor is less than 100 mm³. In oneembodiment, the volume of the tumor is at least 75 mm³. In anotherembodiment, the volume of the tumor is less than 75 mm³. In anotherembodiment, the volume of the tumor is less than 70 mm³. In anotherembodiment, the volume of the tumor is less than 65 mm³. In anotherembodiment, the volume of the tumor is less than 60 mm³. In anotherembodiment, the volume of the tumor is less than 55 mm³. In oneembodiment, the volume of the tumor is at least 50 mm³. In otherembodiments, the tumor is less than 50 mm³. In another embodiment, thevolume of the tumor is less than 45 mm³. In other embodiments, thevolume of the tumor is less than 40 mm³. In another embodiment, thevolume of the tumor is less than 35 mm³. In still another embodiment,the volume of the tumor is less than 30 mm³. In another embodiment, thevolume of the tumor is less than 25 mm³. In still another embodiment,the volume of the tumor is less than 20 mm³. In another embodiment, thevolume of the tumor is less than 15 mm³. In still another embodiment,the volume of the tumor is less than 10 mm³. In still anotherembodiment, the volume of the tumor is less than 12 mm³. In stillanother embodiment, the volume of the tumor is less than 9 mm³. In stillanother embodiment, the volume of the tumor is less than 8 mm³. In stillanother embodiment, the volume of the tumor is less than 7 mm³. In stillanother embodiment, the volume of the tumor is less than 6 mm³. In stillanother embodiment, the volume of the tumor is less than 5 mm³.

In one embodiment, the tumor has a length of at least 5 mm prior tosurgical recision using a PSMA-targeted NIR dye compound of the presentinvention. In one embodiment, these methods detect tumors less than 5mm. In other embodiments the methods herein detect tumors less than 4mm. In some embodiments, the methods herein detect tumors less than 3mm. In another embodiment, the tumor has a length of at least 6 mm. Instill another embodiment, the tumor has a length of at least 7 mm. Inyet another embodiment, the tumor has a length of at least 8 mm. Inanother embodiment, the tumor has a length of at least 9 mm. In stillanother embodiment, the tumor has a length of at least 10 mm. In yetanother embodiment, the tumor has a length of at least 11 mm. In afurther embodiment, the tumor has a length of at least 12 mm. In still afurther embodiment, the tumor has a length of at least 13 mm. In still afurther embodiment, the tumor has a length of at least 14 mm. In anotherembodiment, the tumor has a length of at least 15 mm. In yet anotherembodiment, the tumor has a length of at least 16 mm. In still anotherembodiment, the tumor has a length of at least 17 mm. In a furtherembodiment, the tumor has a length of at least 18 mm. In yet a furtherembodiment, the tumor has a length of at least 19 mm. In still a furtherembodiment, the tumor has a length of at least 20 mm. In anotherembodiment, the tumor has a length of at least 21 mm. In still anotherembodiment, the tumor has a length of at least 22 mm. In yet anotherembodiment, the tumor has a length of at least 23 mm. In a furtherembodiment, the tumor has a length of at least 24 mm. In still a furtherembodiment, the tumor has a length of at least 25 mm. In yet a furtherembodiment, the tumor has a length of at least 30 mm.

In some embodiments the present disclosure relates to prostate specificmembrane antigen (PSMA) targeted compounds conjugated to near-infra red(NIR) dyes and methods for their therapeutic and diagnostic use. Morespecifically, this disclosure provides compounds and methods fordiagnosing and treating diseases associated with cells expressingprostate specific membrane antigen (PSMA), such as prostate cancer andrelated diseases. The disclosure further describes methods andcompositions for making and using the compounds, methods incorporatingthe compounds, and kits incorporating the compounds. It has beendiscovered that a PSMA-targeted compound, such as DUPA or conjugatingPSMA-targeting ligand to an NIR dye via a linker (L) may be useful inthe imaging, diagnosis, and/or treatment of prostate cancer, and relateddiseases that involve pathogenic cell populations expressing orover-expressing PSMA. PSMA is a cell surface protein that isinternalized in a process analogous to endocytosis observed with cellsurface receptors, such as vitamin receptors. PSMA also express in theneo-vasculature of most of solid tumors. Accordingly, it has beendiscovered that certain conjugates that include a linker having apredetermined length, and/or a predetermined diameter, and/orpreselected functional groups along its length may be used to treat,image, and/or diagnose such diseases.

In one illustrative embodiment, the linker L may be a releasable ornon-releasable linker. In one aspect, the linker L is at least about 7atoms in length. In one variation, the linker L is at least about 10atoms in length. In one variation, the linker L is at least about 14atoms in length. In another variation, the linker L is between about 7and about 22, between about 7 and about 24, or between about 7 and about20 atoms in length. In another variation, the linker L is between about14 and about 31, between about 14 and about 24, or between about 14 andabout 20 atoms in length.

In an alternative aspect, the linker L is at least about 10 angstroms(A) in length.

In one variation, the linker L is at least about 15 A in length. Inanother variation, the linker L is at least about 20 A in length. Inanother variation, the linker L is in the range from about 10 A to about30 A in length.

In an alternative aspect, at least a portion of the length of the linkerL is about 5 A in diameter or less at the end connected to the bindingligand B. In one variation, at least a portion of the length of thelinker L is about 4 A or less, or about 3 A or less in diameter at theend connected to the binding ligand B. It is appreciated that theillustrative embodiments that include a diameter requirement of about 5A or less, about 4 A or less, or about 3 A or less may include thatrequirement for a predetermined length of the linker, thereby defining acylindrical-like portion of the linker. Illustratively, in anothervariation, the linker includes a cylindrical portion at the endconnected to the binding ligand that is at least about 7 A in length andabout 5 A or less, about 4 A or less, or about 3 A or less in diameter.

In another embodiment, the linker L includes one or more hydrophiliclinkers capable of interacting with one or more residues of PSMA,including amino acids that have hydrophilic side chains, such as Ser,Thr, Cys, Arg, Orn, Lys, Asp, Glu, Gin, and like residues. In anotherembodiment, the linker L includes one or more hydrophobic linkerscapable of interacting with one or more residues of PSMA, includingamino acids that have hydrophobic side chains, such as Val, Leu, Phe,Tyr, Met, and like residues. It is to be understood that the foregoingembodiments and aspects may be included in the linker L either alone orin combination with each other. For example, linkers L that are at leastabout 7 atoms in length and about 5 Å, about 4 Å or less, or about 3 Åor less in diameter or less are contemplated and described herein, andalso include one or more hydrophilic linkers capable of interacting withone or more residues of PSMA, including Val, Leu, Phe, Tyr, Met, andlike residues are contemplated and described herein.

In another embodiment, one end of the linker is not branched andcomprises a chain of carbon, oxygen, nitrogen, and sulfur atoms. In oneembodiment, the linear chain of carbon, oxygen, nitrogen, and sulfuratoms is at least 5 atoms in length. In one variation, the linear chainis at least 7 atoms, or at least 10 atoms in length. In anotherembodiment, the chain of carbon, oxygen, nitrogen, and sulfur atoms arenot substituted. In one variation, a portion of the chain of carbon,oxygen, nitrogen, and sulfur atoms is cyclized with a divalent fragment.For example, a linker (L) comprising the dipeptide Phe-Phe may include apiperazin-1,4-diyl structure by cyclizing two nitrogens with an ethylenefragment, or substituted variation thereof.

In another embodiment, pharmaceutical compositions are described herein,where the pharmaceutical composition includes the conjugates describedherein in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA. Illustratively, the pharmaceutical compositionsalso include one or more carriers, diluents, and/or excipients.

In another embodiment, methods for treating diseases and disease states,diagnosing diseases or disease states, and/or imaging tissues and/orcells that are associated with pathogenic populations of cellsexpressing or over expressing PSMA are described herein. Such methodsinclude the step of administering the conjugates described herein,and/or pharmaceutical compositions containing the conjugates describedherein, in amounts effective to treat diseases and disease states,diagnose diseases or disease states, and/or image tissues and/or cellsthat are associated with pathogenic populations of cells expressing orover expressing PSMA.

In some embodiments, it is shown herein that such PSMA-targeted NIR dyeconjugates bind to PSMA expressing tumor cells within a tissue.Moreover, the intensity of the fluorescence in greater than theintensity of previously observed with other near infrared dyes that aretargeted with folate for folate receptor positive tumors. This increasedintensity allows the targeting and clear identification of smaller areasof biological samples (e.g., smaller tumors) from a tissue beingmonitored. In addition, the increased intensity of the compounds of thepresent invention provides the added advantage that lowerdoses/quantities of the dye can be administered and still producesmeaningful results. Thus, the compounds of the present invention lead tomore economical imaging techniques. Moreover, there is an addedadvantaged that a lower dose of the compounds of the invention ascompared to conventional imaging compounds minimizes the toxicity andother side effects that are attendant with administration of foreignmaterials to a body.

Furthermore, identification of small tumors will lead to a more accurateand more effective resection of the primary tumor to produce negativemargins, as well as accurate identification and removal of the lymphnodes harboring metastatic cancer cells and identification of satellitedisease. Each of these advantages positively correlates with a betterclinical outcome for the patient being treated.

In specific embodiments, it is contemplated that in addition to tyrosineand tyrosine derivatives, a PSMA-targeted conjugate of a near infrareddye with cysteine or cysteine derivatives also may be useful.Furthermore, it is contemplated that a direct linkage of thePSMA-targeted moiety to the dye or linkage of the dye to DUPA or aPSMA-targeted ligand through an amine linker also produces a loss ofintensity of the fluorescence from the conjugate whereas the presence ofthe tyrosine or tyrosine derivative as the linking moiety betweenenhances the fluorescence of the conjugated compound as a result of thefact that the tyrosine-based compounds of the invention do not requirean extra amine linker to conjugate the SO456 and further becauseconjugation through the phenol moiety of the tyrosine leads to enhancedfluorescence.

The compounds can be used with fluorescence-mediated moleculartomographic imaging systems, such as those designed to detectnear-infrared fluorescence activation in deep tissues. The compoundsprovide molecular and tissue specificity, yield high fluorescencecontrast, brighter fluorescence signal, and reduce backgroundautofluorescence, allowing for improved early detection and moleculartarget assessment of diseased tissue in vivo (e.g., cancers). Thecompounds can be used for deep tissue three dimensional imaging,targeted surgery, and methods for quantifying the amount of a targetcell type in a biological sample.

In specific embodiments, the linker is less than ten atoms. In otherembodiments, the linker is less than twenty atoms. In some embodiments,the linker is less than 30 atoms. In some embodiments, the linker isdefined by the number of atoms separating the PSMA-targeting compoundand the NIR dye. In another embodiment, linkers have a chain length ofat least 7 atoms. In some embodiments, linkers have a chain length of atleast 14 atoms. In another embodiment, linkers have a chain length inthe range from 7 atoms to 20 atoms. In another embodiment, linkers havea chain length in the range of 14 atoms to 24 atoms.

PSMA-targeting compounds suitable for use in the present invention canbe selected, for example, based on the following criteria, which are notintended to be exclusive: binding to live cells expressing PSMA; bindingto neo-vasculature expressing PSMA; high affinity of binding to PSMA;binding to a unique epitope on PSMA (to eliminate the possibility thatantibodies with complimentary activities when used in combination wouldcompete for binding to the same epitope); opsonization of cellsexpressing PSMA; mediation of growth inhibition, phagocytosis and/orkilling of cells expressing PSMA in the presence of effector cells;modulation (inhibition or enhancement) of NAALADase, folate hydrolase,dipeptidyl peptidase IV and/or γ-glutamyl hydrolase activities; growthinhibition, cell cycle arrest and/or cytotoxicity in the absence ofeffector cells; internalization of PSMA; binding to a conformationalepitope on PSMA; minimal cross-reactivity with cells or tissues that donot express PSMA; and preferential binding to dimeric forms of PSMArather than monomeric forms of PSMA.

PSMA-targeting compounds, PSMA antibodies and antigen-binding fragmentsthereof provided herein typically meet one or more, and in someinstances, more than five of the foregoing criteria. In someembodiments, the PSMA-targeting compounds of the present invention meetsix or more of the foregoing criteria. In some embodiments, thePSMA-targeting compounds of the present invention meet seven or more ofthe foregoing criteria. In some embodiments, the PSMA-targetingcompounds of the present invention meet eight or more of the foregoingcriteria. In some embodiments, the PSMA-targeting compounds of thepresent invention meet nine or more of the foregoing criteria. In someembodiments, the PSMA-targeting compounds of the present invention meetten or more of the foregoing criteria. In some embodiments, thePSMA-targeting compounds of the present invention meet all of theforegoing criteria.

Examples of tumors that can be imaged with the PSMA-targeted compoundsof the present invention (e.g., PSMA-targeted NIR dye conjugates)provided herein, include any tumor that expresses PSMA such as, e.g.,prostate, bladder, pancreas, lung, colon, kidney, melanomas andsarcomas. A tumor that expresses PSMA includes tumors withneovasculature expressing PSMA.

In some embodiments, a PSMA-targeted molecules bind to PSMA and areinternalized with PSMA expressed on cells. Thus, a PSMA ligand conjugatecomprising a internalized with PSMA expressed on cells. The mechanism bywhich this internalization occurs is not critical to the practice of thepresent invention.

In some embodiments, the PSMA targeting compounds bind to aconformational epitope within the extracellular domain of the PSMAmolecule. In other embodiments, a PSMA-targeting compound binds to adimer-specific epitope on PSMA. Generally, the compound that binds to adimer-specific epitope preferentially binds the PSMA dimer rather thanthe PSMA monomer. In some embodiments of the present invention, thePSMA-targeting compound preferentially binds to the PSMA dimer. In someembodiments of the present invention, the PSMA-targeting compound has alow affinity for the monomeric PSMA protein.

In some embodiments, the PSMA-targeting compound is a ligand. In someembodiments, the PSMA-targeting compound is2-[3-(1,3-dicarboxypropyl)ureido] pentanedioic acid (DUPA). In someembodiments, the PSMA-targeting compound is DUPA or derivative of DUPA,ligand, inhibitor, or agonist that binds to PSMA-expressing live cells.

The PSMA-targeting NIR dye of the present invention produces atumor-to-background signal ratio that is higher than thetumor-to-background signal ratio of the PSMA-targeting compoundconjugated to a non-NIR dye or non-targeted NIR dye. In someembodiments, the improvement is 10-fold. In some embodiments, thetumor-to-background signal ratio is at least a 4-fold improvement. Insome embodiments, the tumor-to-background ratio is increased by at least1.5-fold. In some embodiments, the PSMA-targeted NIR dye backgroundsignal is half the background signal of the PSMA-targeted compoundconjugated to a fluorescent dye reactive to light less than 600 nm inwavelength. In some embodiments of the present invention, methods usingthe PSMA-targeted NIR dye on live cells produces a background signalless than half the background signal of the PSMA-targeted compoundconjugated to a fluorescent dye reactive to light less than 600 nm inwavelength. In some embodiments of the present invention, methods usingthe PSMA-targeted NIR dye on live cells produces a background signalless than half the background signal of the PSMA-targeted compoundconjugated to a fluorescent dye reactive to light less than 500 nm inwavelength. In some embodiments of the present invention, methods usingthe PSMA-targeted NIR dye on live cells produces a background signalless than one third of the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 600nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one third of the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 500nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one fourth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 600nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one fourth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 500nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one fifth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 600nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one fifth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 500nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one eighth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 600nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one eighth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 500nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one tenth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 600nm in wavelength. In some embodiments of the present invention, methodsusing the PSMA-targeted NIR dye on live cells produces a backgroundsignal less than one tenth the background signal of the PSMA-targetedcompound conjugated to a fluorescent dye reactive to light less than 500nm in wavelength.

In some embodiments, the PSMA-targeting compound is a small moleculeligand that binds specifically PSMA. Such small molecule ligands maybind to the enzymatic site of PSMA in its native conformation. Also,such small molecule ligands may possess any one or more of thecharacteristics for PSMA antibody ligands.

This disclosure also provides methods for synthesizing amino acidlinking groups that are conjugated to a PSMA-targeting compound used forthe targeted imaging of PSMA-expressing cells, tissues, or tumors. Incertain embodiments, this disclosure relates to a compound or a saltderivative thereof, that comprises a PSMA-targeting compound, a linkinggroup, and an NIR dye. In certain embodiments, the linking group can bean amino acid, an isomer, a derivative, or a racemic mixture thereof. Insome aspects, the dye is selected from the group consisting of LS288,IR800, SP054, S0121, KODAK, S2076, S0456 and/or the dyes selected fromgroup consisting of.

In some aspects, this disclosure provides a method of conjugating anamino acid linking group to an NIR dye, wherein the amino acid can betyrosine, serine, theronine, lysine, arginine, asparagine, glutamine,cysteine, selenocysteine, isomers, and the derivatives thereof. Incertain embodiments, the amino acid, isomers, or the derivativesthereof, contain an —OH, —NH₂, or —SH functional group that uponaddition of the fluorescent dye in slight molar excess produces theconjugation of fluorescent group with the amino acid, isomer, or thederivatives thereof. In other embodiments, the amino acid, isomers, orthe derivatives thereof, contains an —OH functional group that uponsynthesis generates an ether bond with the dye that increases thebrightness and detection of the compound. In some embodiments, thisdisclosure relates to the conjugation of the amino acid linking groupwith the NIR dye, wherein the amino acid, isomers, or the derivativesthereof, contains an —SH, —SeH, —PoH, or —TeH functional group that uponsynthesis generates a C—S, C—Se, C—Po, or C—Te bond with the dye. Insome aspects, this disclosure relates to the conjugation of the aminoacid linking group to a dye that has an absorption and emission maximabetween about 500 nm and about 900 nm. In other aspects, the amino acidlinking group is conjugated to a fluorescent dye that has an absorptionand emission maxima between about 600 nm and about 800 nm.

In additional embodiments, this disclosure provides a method forconjugating the amino acid linking group to a PSMA ligand, wherein theamino acid linking group is tyrosine, serine, threonine, lysine,arginine, asparagine, glutamine, cysteine, selenocysteine, isomers orthe derivatives thereof, and is conjugated to folate through a dipeptidebond. In additional aspects, this disclosure provides a method ofconjugating the linking group with a folate ligand, wherein the linkinggroup is tyrosine, serine, threonine, lysine, arginine, asparagine,glutamine, cysteine, selenocysteine, isomers, or the derivativesthereof. In other embodiments, this disclosure relates to a method ofconjugating a pteroyl ligand to an amino acid linking group, wherein thelinking group is tyrosine, serine, threonine, lysine, arginine,asparagine, glutamine, cysteine, selenocysteine, isomers or thederivatives thereof. In certain aspects, the carboxylic acid of thelinking group is bound to the alpha carbon of any amino acid, henceincreasing the specificity of the compound for targeted receptors. Insome embodiments, the charge of the linker contributes specificity tothe compound, wherein the observed binding affinity of the compound totargeted receptors is at least 15 nM.

In other embodiments, this disclosure relates to the use of a compounddesignated, DUPA-EAOA-Tyr-S0456, wherein EAOA is eight aminooctonoicacid, for image guided surgery, tumor imaging, prostate imaging,PSMA-expressing tissue imaging, PSMA-expressing tumor imaging, infectiondiseases, or forensic applications. In other aspects, the compound is aDUPA-EAOA-Tyr-S0456 derivative selected from the group consisting ofDUPA-EAOA-(D)Tyr-S0456, DUPA-EAOA-homoTyr-S0456,DUPA-EAOA-beta-homo-Tyr-S0456, DUPA-EAOA-(NMe)-Tyr-S0456,DUPA-EAOA-Tyr(OMe)-S0456, DUPA-EAOA-Tyr(OBn)-S0456,DUPA-EAOA-NHNH-Tyr-OAc-S0456, salts, and derivatives thereof.

In some embodiments, the PSMA-targeted compound of the present inventionis a small molecule ligand of PSMA.

PSMA-Targeted NIR Dye Conjugates and Their Synthesis

The following schemes show the synthesis of PSMA-targeted NIR dyeconjugates of the present invention.

The examples that follow are merely provided for the purpose ofillustrating particular embodiments of the disclosure and are notintended to be limiting to the scope of the appended claims. Asdiscussed herein, particular features of the disclosed compounds andmethods can be modified in various ways that are not necessary to theoperability or advantages they provide. For example, the compounds canincorporate a variety of amino acids and amino acid derivatives as wellas targeting ligands depending on the particular use for which thecompound will be employed. One of skill in the art will appreciate thatsuch modifications are encompassed within the scope of the appendedclaims.

EXAMPLES Example (1) Pre-Clinical Evaluation of PSMA-Targeted NIR DyeConjugates with Random Variation of Length of the Linker/Spacer Betweenthe Ligand and the NIR Dye

(a) In Vitro Studies.

FIG. 2 shows Structure of PSMA-targeted DUPA-FITC (Fluoresceinisothiocyanate) conjugate (14) and its binding affinity (K_(D)) andspecificity on PSMA-positive 22Rv1 human prostate cancer cells and onPSMA-negative A549 human alveolar basal epithelial cells in culture.DUPA-FITC dissolved in RPMI medium was added at the indicatedconcentrations to 22Rv1 or A549 cells in RPMI culture media and allowedto incubate for 1 h at 37° C. Media was then removed, washed with freshmedia (3x), and replaced with PBS (phosphate buffered saline). Sampleswere analyzed using flow cytometry. Error bars represent SD (n=3). **does not bind to A549 cells

FIG. 3 Relative binding affinities of DUPA-NIR conjugates 1-9 withrespect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancercells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry.

The binding affinity of the DUPA-NIR conjugates was monitored and thedata are shown in Table 1.

TABLE 1 Binding affinity of DUPA-NIR conjugates with variable lengthspacers to PSMA- positive 22Rv1 human prostate cancer cells Number ofatoms between DUPA Compound and NIR agent K_(D) (nM) 1 3 141.9 2 3 112.73 7 9.71 4 12 15.2 5 15 12.2 6 18 35.7 7 22 26.8 8 22 23.2 9 21 17.2

In vivo studies. For in vivo analysis, the tissue distribution ofDUPA-NIR conjugates was monitored and is shown in FIG. 4. Morespecifically, biodistribution of DUPA-NIR conjugates 1-9 was monitoredusing fluorescence imaging of mice bearing human prostate tumorxenografts (22Rv1 cells). Male nude mice with 22Rv1 tumor xenograftswere injected with DUPA-NIR dye conjugates via tail vein. The mice wereeuthanized 2 h after administration of the DUPA-NIR dye conjugate,selected tissues were harvested, and tissues were imaged with IVISimager (ex=745 nm, em=ICG, exposure time=1 s). The results are shown inFIG. 4.

The conjugates were also tested to show the ratio of tumor-to-tissuefluorescence. FIG. 5 shows the tumor-to-tissue fluorescence ratio fromtissue biodistribution data of PSMA-targeted DUPA-NIR conjugates 1-9.After imaging, fluorescence within a Region of interest (ROI) wasmeasured for each tissue using In Vivo imaging software andtumor-to-tissue fluorescence was then calculated

Conclusion: The in vitro binding affinity data showed that the compounds3 (7 atom spacer), 4 (12 atom spacer), and 5 (15 atom spacer) have veryhigh affinity for PSMA whereas the compounds 1 (3 atom spacer) and 2 (3atom spacer) have low affinity for PSMA. The above data show that thePMSA-targeted NIR dye need a minimum length of a 7 atom spacer betweenDUPA and NIR agent to have optimal effective binding affinity.

Compound 4, DUPA-EAOA-Tyr-S0456, (EAOA—Eight aminooctonoic acid) showedthe best tumor-to-background ratio (TBR) out of all compounds evaluated.Compound 4 also showed higher fluorescence intensity in the tumor.Compounds 6 and 7 showed the second and third best TBR amongst thecompound evaluated in this example. However, fluorescence intensity inthe tumor for compound 6 and 7 was lower as compared to that of compound3, 4, and 5. After considering affinity and specificity for PSMAexpressing prostate cancer cells and tumor tissues, fluorescenceintensity in the tumor, tumor-to-background ratio, etc., it appears thatCompound 4 can be considered as a suitable clinical candidate althoughthe other compounds also may provide some valuable insights in theclinic as well as in experimental conditions.

Example 2 Pre-Clinical Evaluation of PSMA-Targeted NIR Conjugates withAromatic Amino Acid Linkers Between the Ligand and the NIR Dye

FIG. 6 shows the structures of PSMA-targeted DUPA-Linker-NIR imagingagents with aromatic amino acid linkers between the ligand and the NIRdye. The synthesis scheme is shown in scheme 3.

-   -   (b) Synthesis

in vitro studies. FIG. 7 shows the Relative binding affinities ofDUPA-NIR conjugates with aromatic amino acids linkers with respect toDUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancer cells wereincubated for 1 h at 37° C. in the presence of 100 nM DUPA-FITC withincreasing concentrations of DUPA-NIR conjugates. Media was thenremoved, washed with fresh media (3x), and replaced with PBS. Cell boundfluorescence was assayed as using flow cytometry.

Table 2 shows data of the binding affinity of DUPA-NIR conjugates witharomatic linkers to PSMA-positive 22Rv1 human prostate cancer cells.

Compound K_(D) (nM) 15 4.9 23 7.5 25 6.3 27 22.2 32 32 33 16 34 34.9 3523.9 36 13.4

in vivo studies. FIG. 8 shows Tissue biodistribution analysis andtumor-to-tissue ratio of DUPA-NIR conjugates 15 and 23 usingfluorescence imaging of mice bearing human prostate tumor xenografts(22Rv1 cells). Male nude mice with 22Rv1 tumor xenografts were injectedwith DUPA-NIR dye conjugates via tail vein. The mice were euthanized 2 hafter administration of the DUPA-NIR dye conjugate, selected tissueswere harvested, and tissues were imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s). After imaging, fluorescence within a Regionof interest (ROI) was measured for each tissue using In Vivo imagingsoftware and tumor-to-tissue fluorescence was then calculated.

FIG. 9 shows an overlay of whole or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 15 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

FIG. 10 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 20 nmol of 23and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

FIG. 11 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 20 nmol of 25and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals

FIG. 12 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 6 nmol of 35and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

FIG. 13 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 6 nmol of 36and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

Conclusion: These in vitro binding affinity data showed that compounds15, 23, 25 and 36 have very high affinity for PSMA. Moreover, compounds15, 23, 25, 35, and 36 showed very good whole-body imaging data within2-4 hours after administering to the animal. In addition, compounds 15and 35 showed excellent tumor-to-background ratio (TBR). Afterconsidering affinity and specificity for PSMA expressing prostate cancercells and tumor tissues, fluorescence intensity in the tumor,tumor-to-background ratio, ease synthesis and availability of startingmaterials for low cost, compound 15 and 35 can be considered asexcellent clinical candidates, although the other compounds also may beuseful both as clinical and/or experimental candidates.

Example (3) Pre-Clinical Evaluation of PSMA-Targeted NIR Conjugates witha Positive Charge Linker Between the Ligand and the NIR Dye

FIG. 14 shows the structures of PSMA-targeted DUPA-Linker-NIR imagingagents with positive charge linkers between the ligand and the NIR dyeand the synthesis scheme for these agents is shown in Scheme 4:

FIG. 15 shows the relative binding affinities of DUPA-NIR conjugateswith respect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostatecancer cells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry.

In vivo studies: FIG. 16 shows the tumor to tissue ratio of DUPA-NIRconjugates 39 and 41 using fluorescence imaging of mice bearing humanprostate tumor xenografts (22 Rv1 cells). Male nude mice with 22Rv1tumor xenografts were injected with DUPA-NIR dye conjugates via tailvein. The mice were euthanized 2 h after administration of the DUPA-NIRdye conjugate, selected tissues were harvested, and tissues were imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s). After imaging,fluorescence within a Region of interest (ROI) was measured for eachtissue using In Vivo imaging software and tumor-to-tissue fluorescencewas then calculated.

FIG. 17 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 20 nmol of 39and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

FIG. 18 shows and overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 20 nmol of 40and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

FIG. 19 shows an overlay of whole body or half body fluorescence imageover white light images after adjusting the threshold. 22Rv1 humanprostate tumor xenograft bearing mouse was injected with 20 nmol of 41and imaged with IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) atdifferent time intervals.

Conclusion: These in vitro binding affinity data showed that thecompound 41 has very high affinity for PSMA. Compounds 39, 40 and 41showed very good whole-body imaging and fast skin clearance in timedependent imaging studies. Adding Arg to the linker between theligand-eight aminooctonoic acid linker and NIR dye, increased the numberof positive charges and decreased the total negative charge of theoverall molecule. Although having Arg moieties decreased the affinity ofthe molecule to PSMA, these compounds showed fast skin clearance. Afterconsidering affinity and specificity for PSMA expressing prostate cancercells and tumor tissues, fast skin clearance, the compound 41 can beconsidered as a clinical candidate, although the other compounds alsomay be useful both as clinical and/or experimental candidates.

Example (4) Pre-Clinical Evaluation of PSMA-Targeted NIR Conjugates witha Negative Charge Linker Between the Ligand and the NIR Dye

FIG. 20 shows Structures of PSMA-targeted DUPA-Linker-NIR imaging agentswith negative charge linkers between the ligand and the NIR dye. Thesynthesis scheme is shown in Scheme 5.

In vitro studies: FIG. 21 Relative binding affinities of DUPA-NIRconjugates of 49 and 50 with respect to DUPA-FITC (14). PSMA-positive22Rv1 human prostate cancer cells were incubated for 1 h at 37° C. inthe presence of 100 nM DUPA-FITC with increasing concentrations ofDUPA-NIR conjugates. Media was then removed, washed with fresh media(3x), and replaced with PBS. Cell bound fluorescence was assayed asusing flow cytometry.

In vivo studies. FIG. 22 shows Tissue biodistribution analysis andtumor-to-tissue ratio of DUPA-NIR conjugates 49 and 50 usingfluorescence imaging of mice bearing human prostate tumor xenografts(22Rv1 cells). Male nude mice with 22Rv1 tumor xenografts were injectedwith DUPA-NIR dye conjugates via tail vein. The mice were euthanized 2 hafter administration of the DUPA-NIR dye conjugate, selected tissueswere harvested, and tissues were imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s). After imaging, fluorescence within a Regionof interest (ROI) was measured for each tissue using In Vivo imagingsoftware and tumor-to-tissue fluorescence was then calculated.

Conclusion: While it had low binding affinity for PSMA, the compound 49has very high tumor accumulation (high fluorescence intensity) and goodtumor-to-background ratio

Example (5) Pre-Clinical Evaluation of PSMA-Targeted NIR Dye Conjugateswith Variation of Charge of the NIR Dye Molecule

FIG. 23 shows structures of PSMA-targeted DUPA-Linker-NIR imaging agentswith variably charged NIR dye molecule.

FIG. 24: Relative binding affinities of DUPA-NIR conjugates with respectto DUPA-FITC (14). PSMA-positive 22Rv1 human prostate cancer cells wereincubated for 1 h at 37° C. in the presence of 100 nM DUPA-FITC withincreasing concentrations of DUPA-NIR conjugates. Media was thenremoved, washed with fresh media (3x), and replaced with PBS. Cell boundfluorescence was assayed as using flow cytometry.

Compound K_(D) (nM) 15 4.9 54 2.6 55 7.3 56 3.7 59 60.9 60 5.4

FIG. 25: Overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 54 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

FIG. 26 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 55 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

FIG. 27 shows Overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 56 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

FIG. 28 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 57 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals

FIG. 29 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 58 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals

FIG. 30 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 60 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

Conclusion: These in vitro binding affinity data showed that thecompounds 15, 55, 56, and 60 have very high affinity for PSMA. Compounds15, 54, 57 and 60 showed very good whole-body imaging and fast skinclearance in time dependent imaging studies. Therefore, reducingnegative charge by removal of sulfonic acid groups (SO3H) from the NIRdye helped in producing fast skin clearance and fast tumor accumulation.After considering affinity and specificity for PSMA expressing prostatecancer cells and tumor tissues, fast skin clearance, the compounds 54,57, and 60 can be considered as clinical candidates.

Example (6) Pre-Clinical Evaluation of PSMA-Targeted NIR Dye Conjugates:Miscellaneous DUPA-NIR Conjugates

FIG. 31: Structures of PSMA-targeted DUPA-Linker-NIR imaging agents withmiscellaneous linkers and NIR dyes.

FIG. 32 shows the relative binding affinities of DUPA-NIR conjugateswith respect to DUPA-FITC (14). PSMA-positive 22Rv1 human prostatecancer cells were incubated for 1 h at 37° C. in the presence of 100 nMDUPA-FITC with increasing concentrations of DUPA-NIR conjugates. Mediawas then removed, washed with fresh media (3x), and replaced with PBS.Cell bound fluorescence was assayed as using flow cytometry

Compound K_(D) (nM) 62 141.4 64 318.8 65 983 66 Not active

in vivo studies. FIG. 33 shows overlay of whole body or half bodyfluorescence image over white light images after adjusting thethreshold. 22Rv1 human prostate tumor xenograft bearing mouse wasinjected with 20 nmol of 63 and imaged with IVIS imager (ex=745 nm,em=ICG, exposure time=1 s) at different time intervals.

FIG. 34 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 6 nmol of 63 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

FIG. 35 shows Overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 20 nmol of 64 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

Conclusion: These in vitro binding affinity data showed that thecompounds 62, 64, 65, and 66 have low affinity for PSMA. However,Compounds 63 and 64 also showed very good whole-body imaging and fastskin clearance in time dependent imaging studies. Therefore, thecompounds 63 and 64 can be considered as particularly preferred clinicalcandidates, although the other compounds also may be useful both asclinical and/or experimental candidates.

Example (7) Pre-Clinical Evaluation of PSMA-Targeted NIR Dye Conjugates:Alternative Ligands for DUPA

FIG. 36 shows Structures of PSMA-targeted NIR imaging agents withdifferent ligand.

FIG. 37 shows relative binding affinities of PSMA-targeted NIRconjugates 15 with respect to DUPA-FITC (14) for PSMA-positive 22Rv1 andfor PSMA-negative A549 cells. Cancer cells were incubated for 1 h at 37°C. in the presence of 100 nM DUPA-FITC with increasing concentrations ofcompound 15. Media was then removed, washed with fresh media (3x), andreplaced with PBS. Cell bound fluorescence was assayed as using flowcytometry.

FIG. 38 shows overlay of whole body or half body fluorescence image overwhite light images after adjusting the threshold. 22Rv1 human prostatetumor xenograft bearing mouse was injected with 6 nmol of 15 and imagedwith IVIS imager (ex=745 nm, em=ICG, exposure time=1 s) at differenttime intervals.

Conclusion: While alternative ligands for DUPA that have higher affinityfor PSMA when compared to DUPA have been synthesized, this example showsthat the compound 15 has a very high affinity for PSMA-positive 22Rv1cells but not for PSMA-negative A549 cells indicating the compound 15 ishighly specific for PSMA. Time dependent whole-body imaging studiesshowed that the compound 15 accumulated in PSMA-positive tumors andkidneys of the mouse, again demonstrating that compound 15 is anexcellent clinical candidate.

The invention claimed is:
 1. A compound having a formula: B-X-Y-Z,wherein B comprises DUPA or a derivative thereof; X is EAOA, Y isselected from the group consisting of phenylalanine-tyrosine,histidine-tyrosine, phenylalanine-arginine-tyrosine, andhistidine-tyrosine; and Z comprises S0456.
 2. A compound selected fromthe group consisting of:


3. The compound of claim 1 wherein the compound has an absorption andemission maxima between about 500 nm and about 900 nm.
 4. The compoundof claim 1 wherein the compound is made to fluoresce after distributionthereof in tissue cells.
 5. The compound of claim 4, wherein the tissuecells are selected from the group consisting of prostate cells, prostatecancer cells, bladder cancer cells, pancreatic cancer cells, livercancer cells, lung cancer cells, kidney cancer cells, sarcoma cells,breast cancer cells, brain cancer cells, neuroendocrine carcinoma cells,colon cancer cells, testicular cancer cells and melanoma cells.
 6. Amethod of optical imaging of a biological tissue that expresses PSMA,the method comprising: (a) contacting the biological tissue with acomposition of claim 1, (b) allowing time for the compound in thecomposition to distribute within a biological target; (c) illuminatingthe tissue with an excitation light of a wavelength absorbable by thecompound; and (d) detecting an optical signal emitted by the compound.7. The method of claim 6, wherein the optical signal emitted by thecompound is used to construct an image.
 8. The method of claim 6,wherein the biological tissue is selected from the group consisting ofprostate cells, prostate cancer cells, bladder cancer cells, pancreaticcancer cells, liver cancer cells, lung cancer cells, kidney cancercells, sarcoma cells, breast cancer cells, brain cancer cells,neuroendocrine carcinoma cells, colon cancer cells, testicular cancercells and melanoma cells.
 9. A method of identifying a target cell typein a biological sample comprising a) contacting the biological samplewith a compound of claim 1 for a time and under conditions that allowfor binding of the compound to at least one cell of the target celltype; and b) optically detecting presence or absence of the compound ofin the biological sample, c) wherein presence of the compound indetecting step b) indicates that the target cell type is present in thebiological sample.
 10. The method of claim 6, wherein the optical signalis detected using an endoscope, catheter, tomographic system, hand-heldoptical imaging system, surgical goggles, or intra-operative microscope.